Arch. Biol . Med. E x p . 13, 2 1 3 - 2 3 2 , 1 9 8 0

The Enzymology of DNA Replication

La enzimología de la replicación de DNA

R A F A E L V I C U N A

Laboratorio de Bioquímica. Instituto de Ciencias Biológicas. Universidad Católica de Chile. Casilla 1 14-D. Santiago, Chile.

(Recibido el 26 de marzo de 1980)

I. INTRODUCTION 213

2. T H E INITIATION REACTION 214 2.1. E. dili priming proteins

—dna G gene product —RNA polymerase

2.2. Bacteriophage-coded DNA primases — 1 4 DNA primase —T7 gene 4 protein

2.3. Other primases 2.4. Initiation by a site-specific endonucleolytic

cut — 0 X 174 gene A and fd gene II proteins

3. T H E ELONGATION REACTION 217 3.1. E. coli DNA polimerases

—E. coli DNA polymerase I —E. cob DNA polymerase 111

3.2. Bacteriophage-coded DNA polymerases — T 4 gene 43 protein —T7 gene 5 protein

3.3. DNA polymerases a, B and 7 from eukaryo­tic cells

4. SEALING OF DNA FRAGMENTS 219

4.1. E. coli DNA ligase 4.2. Bacteriophage T 4 gene 30 protein 4.3. Mammalian DNA ligases I and II

C O N F O R M A T I O N A L CHANGES OF DNA DURING REPLICATION 220 5.1. E. coli HDP 5.2. Bacteriophage-coded HDPs

— T 4 gene 32 protein —T7 HDP —fd gene V protein

5.3. Eukaryotic HDPs 5.4. Bacterial DNA topoisomerases

—Swivelases —DNA gyrase

5.5. Bacteriophage-coded DNA topoisomerases — T 4 DNA topoisomerase —Lambda int gene product — 0 X 1 7 4 gene A and fd gene II proteins

5.6. Eukaryotic DNA topoisomerases 5.7. E. coli DNA helicases

—DNA helicase I —DNA helicase II —rep protein —rec BC nuclease

5.8. Bacteriophage-coded DNA helicases — T 4 gene dda protein —T7 gene 4 protein

5.9. Eukaryotic DNA helicases CONCLUSIONS 227 REFERENCES '227 APPENDIX 232

1. INTRODUCTION

Progress made dur ing the last years in our knowledge o f D N A repl ica t ion has shown that it is a somewhat complex process (for recent reviews see refs 1-6). At tempts to elucidate the mechanism o f D N A synthesis have been under taken using simple templates , such as bacterial and animal viral ch romosomes and plasmids. All these systems

have shown that several proteins a re required to initiate and e longate D N A chains, as well as to br ing about the topological changes the D N A molecule must unde rgo dur ing its duplication. T h e requ i remen t for these proteins can be directly demons t ra ted with the utilization o f thermosensi t ive mutants , by using specific inhibitors and by reconsti tut ion o f an in vitro replication system with the purified componen t s . Al though in some cases

'The abbreviations used are: ss: single stranded; ds: double stranded; NEM: N-ethyimaleimide; DNA pol: DNA polymerase; M. W.: molecular weight; ddTTP: 2'3' dideoxythymidine triphosphate; HDP: helix destabilising protein; RF: replicative form; EF: elongation factor; SDS: sodium dodecyl sulfate.

2 1 4 VICl.'ÑA

direct implication of certains proteins has not

been possible, the catalytic activity o f some

proteins (e.g. topoisomerases , helicases)

justify the assumption that they play an

essential role in DNA replication.

T h i s article does not intend to be a critical

nor a comprehens ive review on D N A

synthesis. It represents ra ther a survey of

some o f the proteins that are involved in this

process, both in prokaryot ic and eukaryot ic

systems.

2. T H E INITIATION REACTION

None o f the viral, bacterial o r animal D N A

polymerases isolated to date are able to start

D N A chains de novo, suggesting that o ther

proteins a re involved in chain initiation.

T h e s e enzymes, called D N A primases, show a

high degree o f site specificity when they act at

the origin o f ch romosomal replication. T h e v

must also copy the small ol igonucleot ides that

serve as pr imers for the synthesis o f Okazaky

fragments .

2.1. E. Coll PRIMING PROTEINS

— dna G gene product

Using the ss ' D N A o f phage G 4 as template ,

Bouché et al. (7) were the first to show that the

dna G gene product oi E. coli, in the presence

o f binding protein, catalyzes the synthesis o f a

short polynucleotide p r imer at the origin o f

replication. Later , S. Wickne r (8) demonst ra ­

ted that dna G polymerizes r N T P s and d N T P s

interchangeably, to yield ribo-, deoxyr ibo-

and mixed r ibo-deoxyr ibonucleot ide pr imers .

T h e isolation o f dna G primase has been

approached in three different ways (9 , 10).

O n e m e t h o d c o n s i s t s i n a n in vitro complementa t ion assay, in which dna G

protein present in an ext rac t p repared from

wild type cells restores the activity o f an

ext rac t p repared from a dna G ts mutant. A

second method has been to follow directly the

enzymatic activity o f dna G protein, that is, the

synthesis o f a p r imer using D N A binding

protein plus G 4 , o¡ 3 o r S T - 1 DNAs as

templates. A third method involves the

coupl ing o f the pr iming reaction with the

D N A elongat ion system. In this assay d N M P

incorporat ion is measured , since il is

proport ional to the extent of pr iming.

The dna G protein is a single polypeptide,

(M.W. 6 0 , 0 0 0 ) ; it has an isoelectric point of f>.9

at 8°C and it is resistant to NKM and

rifampicin (9 , 10), but it is sensitive to the

nucleotide analogue 2 ' -deoxy-2 ' -a / ido-

cytidine t r iphosphate ( 1 1 ) .

dna G pr imase recognizes a unique region

at the origin o f minus strand replication o f

phages G 4 , a 3, S T - 1 and 0 K . These various

initiation regions have been located with the

aid o f restriction enzymes and have been

defined at the nucleot ide level by compar ing

the sequence o f these origin sites with the-

sequence o f an R N A pr imer synthesized bv

dna G (12 , 13) . D N A sequencing studies show

that all these origins are very similar, as

compared to adjacent coding regions. It is

possible to a r range them in similar pat terns of

two or three hairpin loops in the regions o f

negative strand initiation suggesting their im­

portance as recognit ion signals for dna G pro­

tein.

Several lines of evidence show that the

main tenance o f the s t ructure at the origin site

ra ther than the sequence itself is essential for

initiation: a) inactive phage D N A can be

activated by heat ing and slow cooling in

the presence of E. coli b inding protein

(9) ; b.) E. coli dna topoisomerase I. an

enzyme known to form knotts with ss DNA

(14 ) , when present , inhibits the p reming

reaction dependent on dna G (9) ; c) three

well separated groups o f nucleot ides within

the negative strand origin o f phage 0 K are

protected by dna G protein agains nuclease

digestion, suggesting some degree o f folding

o f the D N A in a tertiary s tructure ( ) . Sims, E.

Benz , personal communica t ion ) ; d) the

pr iming react ion with 0 X 1 7 4 ss D N A , in

addition to dna G and binding protein,

requires the products o f dna B and dna C, and

the replication factors X , Y and Z (also called i,

n and n') ( 1 5 , 16) . T h e role o f these additional

proteins, which form a pre-pr iming complex

with 0 X 1 7 4 D N A , is perhaps to crea te a site

on the template that can be recognized bv dna

G primase. However , the origin o f 0 X 1 7 4

complementa ry strand synthesis seems not be

unique: i f the pr iming react ion is uncoupled

from D N A synthesis, it gives rise to many

ENZYMOLOGY OF DNA REPLICA I ION 2 1 5

pr imers in each circle ( 1 7 ) . R o m b e r g has proposed (18) that the dna B protein (M.W. 2 8 0 , 0 0 0 ) , aided by its own r N T P a s e action (19 , 2 0 ) , moves processively on the template strand, acting as a mobil replicat ing promoter , dna B protein interacts specifically with dna C protein (M.W. 3 0 , 0 0 0 ) in the presence o f A T P (21) and it is thus t ransferred to the pre-pr iming complex with the help o f protein X (M.W. 4 5 , 0 0 0 ) . (3 ) . Protein Y (M.W. 7 0 , 0 0 0 ) , like dna B , is a DNA-dependen t ATPase or d A T P a s e , but works best with DNAs that require this protein for initiation ( 2 2 ) . T h i s mult ienzyme system o f the host used by 0 X 1 7 4 to pr ime D N A synthesis is probably used by the cell to replicate its own ch romosome .

T h e length o f the p r imer synthesized by dna G in the presence o f G 4 or ct3 D N A as templates and the four r ibonucleot ides is o f 2 8 nucleotides (9 , 2 3 ) . However , the presence o f deoxyr ibonucleot ide residues in the chain limits p r imer chain length (9 , 2 4 ) . This effect occurs even when the r ibonucleot ides are present in a ten-fold excess in the pr iming reaction. Only after p ro longed incubation periods is the full lenght p r imer obtained under these condit ions (9) . W h e n only the four d N T P s are present in the reaction, the rate o f p r imer format ion is ten-fold slower compared to the rate with the four r N T P s alone, and the po lydeoxynucleo t ide chains obtained are he te rogeneous in size. P r imer synthesis is initiated with A T P or d A T P at the 5' end. ( 2 4 ) . I f A D P is used in place o f any o f these nucleoside t r iphosphates , it is incorpora ted both internally and at the 5 ' end (9) .

T o date, several genomes have been shown to require dna G for replication. These include the ss D N A phages, the E. coll. c h r o m o s o m e , plasmid col El and phage lambda.

— RNA polymerase.

Init iat ion o f D N A replication by R N A polymerase both in vivo and in vitro is well documen ted for the Col El type plasmids and the filamentous ss D N A phages id and M 1 3 . In both cases, R N A polymerase initiates D N A synthesis at un ique origin sites and it is not

involved in the pr iming o f Okazaki f ragments . Using fd D N A as template , and in the

presence of binding protein, RNase H (an enzyme that degrades RNA when hvbridized to D N A ) and discriminatorv factors a and B. R N A polymerase recognizes a specific hairpin s tructure o f the viral g e n o m e and transcribes from it a short R N A fragment ( 2 5 - 2 8 ) . This pr imer is subsequently e longated by E. coli D N A polymerase I I I plus the e longat ion factors. The requ i rement o f additional proteins besides R N A polymerase to initiate the pr iming reaction is related to the specificity of the system, i .e. to start at a unique origin and also to avoid unspecific pr iming by R N A polymerase on o the r templates.

T o initiate replication of p B R 3 4 5 , a Col El-type plasmid, R N A polymerase synthesizes a 100 nucleot ide long transcript in a region which is about 4 5 0 bases pairs appar t from the replication origin ( 2 9 ) . Apparent ly , this R N A segment is processed by RNase I I I and RNase H (30 , 3 1 ) and thereaf ter it hybridizes to the origin region where it serves as a p r imer for D N A synthesis. Tomizawa et al. ( 32 ) have mapped the origin o f Col E l D N A replication at the nucleot ide level. T h e y have also found that early D N A fragments (6 S) contain r ibonucleo t ide-DNA linkages, most of them having very few r ibonucleot ides at the i r 5 ' ends ( 3 3 ) .

R N A polymerase is also required to initiate new rounds of replication o f E. coli. c h r o m o s o m e (34 , 3 5 ) . T h e r e is no evidence, however, that the R N A transcr ibed at the origin (36) functions as a p r imer for D N A synthesis. Alternatively, t ranscript ion may be a mechanism by which R N A pol­ymerase activates the origin by separat ing both D N A strands, in o r d e r to help dna G to prime replication in the p resence o f the gene products o f dna A, dna B , dna C, dna I , dna J , dna K and dna P ( 3 7 ) .

Transcr ip t ion by R N A polymerase at t h e i n i t i a t i o n r e g i o n o f p h a g e l a m b d a c h r o m o s o m e has also been shown to be essential for D N A replication, not only to provide the messenger o f genes O and P, but also to activate the origin ( 3 8 ) . Alson in this case, there is no evidence that the t ranscr ipt is elongated by D N A polymerase .

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2.2. BACIERIOPHAGE-CODEl) DNA PRIMASES

— 14 DNA prima.se.

T w o phage T 4 - c o d e d proteins a re required to

initiate replication on a ss D N A template .

T h e y are the product of gene 41 ( 3 9 - 4 0 ) and

another protein tentatively identified as the

product of the DNA-delay gene 61 (40 ) .

Originally, the requ i rement of the latter

protein had been missed because it was

present as a minor contaminant in gene 32

protein preparat ions ( 4 1 ) . G e n e 41 protein

has been purified to nea r homogenei ty (42 ,

4 3 ) . It is a single polypeptide chain with a

M.W. o f 5 8 , 0 0 0 and catalyzes a ss

DNA-dependent hydrolysis o f purinic

nucleoside t r iphosphates . Synthesis o f the

primers, which are 6 to 8 nucleotides long, has

an absolute requ i rement for A T P and G T P .

In contrast to dna G and R N A polymerase,

but similar to T 7 primase, initiation by T 4

proteifis does not requi re b inding protein.

T h e reason for the involvement o f two

proteins in the pr iming react ion o f

bac ter iophage T 4 D N A replication is not

known. It could be related to mechanis t ic

problems inheren t to initiating synthesis in

the lagging strand ( 3 9 ) . I t may also have to do

with the main tenance o f the duplex s t ructure

between the R N A pr imer and the D N A

template , in o r d e r to facilitate elongat ion by

the mul t ienzyme complex fo rmed by proteins

coded by genes 3 2 , 4 3 , 4 4 / 6 2 and 4 5 (39 , 4 4 ) .

—T7 gene 4 protein

G e n e 4 protein o f bac te r iophage T 7 has been

identified as a D N A primase ( 4 5 - 4 8 ) . After

purification with the use o f an in vitro complementa t ion assay, the enzyme has a

M.W. o f 5 8 , 0 0 0 . ( 4 5 , 4 9 ) . G e n e 4 protein, in

the presence o f any natural ss D N A , A T P and

G T P , or a mixture o f all four r N T P s , catalyzes

the s y n t h e s i s o f s h o r t o l i g o n u c l e o t i d e s ,

p r e d o m i n a n t l y p p p A G C A ( 4 5 - 4 8 ) . T h e

primary structure o f these pr imers has been

conf i rmed by T . Okazaki (50 ) by studies done

in vivo. T 7 D N A pol can utilize p p p A C G A o r

synthet ic tri , t e t ra o r p e n t a n u c l e o t i d e s as

chain initiators in the presence o f T 7 gene 4

protein. Apparent ly , in this case the pr imase

plays an active role in chain e longat ion by

stabilizing the shor t p r imer segments in a

duplex state with the D N A template ( 5 1 ) . The

interaction between both T 7 - c o d e d enzymes

is speci f ic , s ince o t h e r D N A p o l y m e r a s e s

cannot replace T 7 DNA pol in the extension

o f t h e s e o l i g o n u c l e o t i d e s . U n l i k e o t h e r

primases, T 7 gene 4 protein is activated five

fold when d N T P s are present , a l though there

is no evidence that they are incorpora ted (45 ) .

An exp lana t ion for this s t imula tory effect

could be that d N T P s protect r N T P s against

hydrolysis by T 7 pr imase, an activity that is

intrinsic to the enzyme. (See also section 5 .8 . ) .

2.3. O T H E R PRIMASES

E. Lanka et al. (52) have recently isolated a

D N A primase specified by the I-like plasmids

( R 6 4 , Gol I ) . T h e enzyme has a sedimentat ion

coefficient o f 3 .6 s and utilizes all four

r ibonucleoside t r iphosphates . In react ions

carr ied out with c rude extracts p repared from

thermosensi t ive dna mutans, this pr imase can

substitute for the host functions of dna B-dna

C-dna G, R N A polymerase and dna G, in the

conversion o f t h e s s D N A o f phage 0 X 1 7 4 , fd

and G 4 to the duplex form, respectively.

So far, information about this enzyme is

rather prel iminary. Data regard ing direct

measurements of R N A synthesis, require­

ments o f the reconst i tuted react ion and

site specificity o f initiation must be

awaited.

Polyoma nascent D N A strands synthesized

in vitro contain decar ibonucleot ides at tached

at their 5 ' end ( 5 3 ) . T h e s e pr imers , called

iRNA, begin always with A T P o r G T P and are

t ranscr ibed randomly on the genome . iRNA

synthesized unde r condit ions o f limiting

r N T P contains d N T P s . T h e initiating enzyme

has been t e rmed primase, in analogy to the

dna G protein of E. coli.

2.4. INITIATION BY A SITE-SPECIEIC

ENDONUCLEOLYTIC C U T

— 0174 gene A and fd gene II products

Circular ds genomes which replicate through

the roll ing circle mechan ism must suffer an

EN/.YMOI.OGY OK l)N A REPLICATION 2 1 7

endonucleolvt ic cleavage in one of their strands in o rde r to unwind the duplex and initiate D N A synthesis. S o m e of the best known examples of this type of replication are those represen ted by phages 0 X 1 7 4 and fd. During the life cycle of these phages, their ss circular c h r o m o s o m e becomes a duplex r ing which multiplies itself several fold utilizing the rolling circle model . ( 5 6 - 5 8 ) .

The 0 X 1 7 4 gene A codes for a site-specific endonuclease called A protein (M.W. 5 8 . 0 0 0 ) which int roduces a ss discontinuity in the viral strand of supercoi led 0 X R F I D N A ( 5 9 - 6 5 ) . T h e location of this nick is between nucleotides 4 2 9 7 and 4 2 9 8 in the A cistron ( 6 6 ) . T h e enzyme is highly specific: it will not cleave re laxed 0 X R F T V , Col El, PM2 , fd o r M 1 3 DNAs ( 6 0 , 6 f ) . After cleavage, the A protein remains covalently at tached to the 5 ' end ( 6 0 , 6 1 ) , while the 3 ' hydroxyl genera ted serves as a p r imer for the synthesis of viral strands. T h e 0 X R F I I D N A A protein complex can be isolated and it is able to support D N A synthesis by itself when supplemented with c rude extracts o f uninfected E. coli ( 6 0 ) .

T h e 0 X A protein does not act catalytically in the cleavage o f 0 X R F I D N A . U n d e r condit ions leading to the quantitative cleavage o f 0 X R F I D N A , the molar ratio of 0 X R F I D N A to added 0 X A protein is approximate ly 1:10, a l though the actual s toichiometry o f the react ion is not known ( 6 1 ) .

E isenberg et al. (64)have repor ted that after the synthesis o f a 0 1 7 4 unit lenght viral s t rand is comple ted , the A protein bound at the 5 ' end ligates the two ends of the viral D N A to form covalently closed circles.

Bac t e r iophage fd gene I I protein is also a site-specific endonuc lease which cleaves the viral s t rand o f fd R F I D N A between nucleotides 5 7 6 3 and 5 7 6 4 ( 6 7 - 7 1 ) . T h i s site is only 2 4 nucleot ides away from the origin of complemen ta ry s trand synthesis, in the integenic region between genes I I and I V ( 7 1 ) . G e n e I I protein has a M.W. o f 4 5 , 0 0 0 , and in contras t to 0 X 1 7 4 A protein, it does not remain bound to the 5 ' end o f the viral s trand. Ins tead , it leaves 3 ' hydroxyl and 5 ' phospha te te rmini ( 7 1 ) . G e n e I I protein is conceivably involved in the unwinding o f the strands dur ing replication and the closing o f

the viral strand which is displaced from the rolling circle ( 6 8 ) .

It can be predicted that replication of phages lambda (72) and PM2 ( 7 3 ) , which also proceed utilizing the roll ing-circle system, will require initiation by site-specific viral-coded nucleases, as yet unidentif ied.

3. T H E ELONGATION REACTION

D N A polymerases are the enzymes that actually synthesize the new polynucleot ide chains. T o accomplish this react ion, they function as one o f the componen t s o f the " repl isome" complex , o f which primases, b inding proteins, e longat ion factors, topoisomerases and helicases are also consti tuents. D N A polymerases are widely distr ibuted in nature , be ing present in bacteria, plant and animal cells. T h e y are also coded by some bacterial and animal viral genomes .

Gene t ic and biochemical evidence have demons t ra ted the presence o f three dif ferent D N A polymerases in the bacter ia E. coli ( 74 , 7 5 ) , B. subtilis ( 76 , 77)and A. calcoaceticus ( 7 8 ) , some o f which have been purif ied to homogenei ty ( 7 9 - 8 2 ) . O n the o the r hand, only one D N A polymerase species has been identified and character ized from M. Luteus (83 ) , T. aquaticus ( 84 ) and the mar ine Pseudomonas BAL-31 ( 8 5 ) .

Eukaryot ic cells contain three D N A polymerases, usually re fe r red to as D N A polymerases a , 3 and 7 (reviewed in refs. 8 6 - 8 8 ) . W h e n they are ex t rac ted using non aqueous solvents, they can be found mainly in the nuclei , a l though D N A pol y is also present in the mi tochondr ia . T h e y all share the c o m m o n proper ty of being devoid o f associated exonuclease activities.

Selec ted features of some D N A polymerases are presented below.

3.1. E. COLI DNA POLYMERASES

— E. coli DNA polymerase I

T h i s enzyme is a single polypeptide o f M.W. 1 0 9 , 0 0 0 and it is resistant to N E M . It represents the most extensively studied of all D N A polymerases. D N A pol I participates

2 1 8 VICUÑA

actively in the repair o f lessions suffered by

the ch romosome . However , its role in D N A

synthesis as an elongat ion enzyme is very

restricted. Only the Col El-type plasmids

requi re D N A pol I for replication, a l though in

conjuct ion with D N A pol I I I ( 89 , ' 2 9 ) .

Nevertheless, the activity o f nick translation

which is intrinsic to D N A pol I (90) has been

shown to be essential for cell viability ( 9 1 ) . The

evidence available (91) suggests that this

activity is in charge o f the processing o f the

pr imers o f the Okazaki f ragments , reaction

after which D N A pol I leaves a nick that is

thereaf ter sealed by D N A ligase.

— E. coli DNA polymerase III

T h i s enzyme.is coded by the gene dna E ( 9 2 ) ,

being the M.W. o f the native enzyme 1 8 0 , 0 0 0 .

It is composed o f subunits o f 1 4 0 , 0 0 0 , 2 5 , 0 0 0

and 1 0 , 0 0 0 (62 , 8 1 ) . In addit ion o f being the

enzyme that replicates the host c h r o m o s o m e ,

D N A p o l I I I a l s o is r e q u i r e d f o r t h e

replication o f the icosahedral and f i lamentous

ss D N A phages , p h a g e l a m b d a and some

plasmids. As D N A pol I , the enzyme contains

both a 5' to 3' and a 3 ' to 5 ' (proofreading)

exonuclease activities (62 , 8 1 , 9 3 ) .

D N A pol I I I is unable to copy by itself a

long pr imed ss D N A template . T o accomplish

this react ion, it requires additional e longat ion

factors ( E F ) and A T P o r d A T P (3 ) . T h e s e

proteins a re : E F I ( M . W . 4 0 , 0 0 0 ) , E F I I I (M.W.

6 3 , 0 0 0 ) and the product o f the gene dna Z

(M.W. 1 1 0 , 0 0 0 ) . T h e mechan i sm o f the

e longat ion react ion has been solved ( 9 4 ) : a

complex fo rmed spontaneously by dna Z

protein and E F I I I catalyzes the t ransfer o f

E F I to a pr imed- templa te in an A T P or

d A T P - d e p e n d e n t react ion. D N A pol I I I

binds then to the E F I D N A template complex

and catalyzes the incorporat ion o f d N T P s into

D N A . T h e fate o f A T P in the react ion is not

clear , since hydrolysis o f this molecule has not

been detected. It might be requi red to induce

a conformat iona l change in one o f these

e longat ion proteins .

R o m b e r g ' s g roup has isolated a different

form o f D N A pol I I I , t e rmed D N A pol I I I

ho loenzyme ( 9 5 , 9 6 ) . T h i s form appears to be

a complex o f the th ree polymerase subunits

plus the E F . T h e identity o f the ho loenzyme

as such is not yet clear , since the E F can be separated from D N A pol I I I by convent ional

ch romatograph ic procedures . In addition,

these E F can also activate E. coli D N A pol II

(97 ) and B. subtilrs D N A pol I I I ( 8 2 ) .

Lately, th ree new proteins, have been

implicated in the e longat ion react ion. O n e o f

them has been called protein u ( 9 6 ) . The

other two, which have been identified

independent ly (96 , 9 8 ) , copurify with D N A

pol I I I and weigh about 8 0 , 0 0 0 , indicating

that they may be the same entity.

T h e part icipation o f at least six and possibly

nine polypeptide chains in the e longat ion o f a

pre-pr imed template , makes this react ion far

m o r e compl ica ted than first imagined.

3.2. BACTERIOPHAGE-CODED DNA

POLYMERASES

— T4 gene 43 protein

T 4 induced D N A pol is a single polypept ide 'of

M . W . 1 1 0 , 0 0 0 . Coded by gene 4 3 , T 4 D N A

pol has a 3 ' to 5 ' exonuc lease activity and it is

inhibited by sulfhydryl blocking reagents

( 9 9 - 1 0 0 ) . T h e prefe r red template for the

enzyme is primed-ss D N A . T h e elongat ion

react ion can be activated specifically by T 4

gene 32 binding protein, which forms a

complex with the polymerase ( 1 0 1 ) .

Incorpora t ion o f d N T P s is also activated

synergistically by the products o f T 4 genes 4 5

and 4 4 / 6 2 , in a react ion that requires A T P

( 1 0 2 , 103 ) . T h e 4 4 / 6 2 protein complex is an

A T P a s e and the mechan ism by which it

increases the efficiency o f the D N A pol

react ion is not known.

— T7 gene 5 protein

T 7 D N A pol is d imer composed o f T 7 gene 5

protein (M.W. 8 4 , 0 0 0 ) and t h i o r e d o x i n ( M . W .

1 2 , 0 0 0 ) , a prote in specified by the host gene

T s n C ( 1 0 4 ) . T h e precise role o f th ioredoxin

in T 7 D N A replicat ion is not known, a l though

it could catalyze r ibonucleot ide reduct ion at

the polymerizat ion site. T 7 D N A pol, as o the r

D N A polymerases o f prokaryot ic origin

contains an intrinsic 3 ' to 5 ' exonuc lease

activity.

ENZYMOl.OGY OK 1)NA RKI'I.ICA I ION 2 1 9

3.3 D N A P O L Y M E R A S E S a, B A N D y F R O M

E U K A R Y O T K ; C E L L S

T h e as ignement of precise M.W. for D N A pol a has been difficult because it exhibits some heterogenei ty . Multiple forms could represent proteolytic degradat ion ( 1 0 5 ) , contaminat ion o f the cells used to isolate the enzyme ( 1 0 6 ) o r the format ion o f complexes between D N A pol a and o the r proteins ( 1 0 7 , 108 ) . Most repor ts est imate the M.W. for D N A pol a ranging from 1 3 0 , 0 0 0 to 1 6 0 , 0 0 0 , with variable subunit composi t ions. T h e enzyme is inhibited by N E M and by NaCl concent ra t ions above 2 5 m M . It exhibits optimal activity with gapped D N A as template and shows little activity with pr imed-r ibohomopolymer templates such as poly A-oligo d T .

D N A pol 3 has a M.W. o f about 4 0 , 0 0 0 and it is resistant to N E M . It is st imulated by 1 0 0 - 2 0 0 m M NaCl and utilizes equally well activated D N A and pr imed-polyr ibo-or polydeoxyribonucleot ides as templates.

D N A pol 7 comprises about 1% o f the total cellular polymerase activity. T h e M.W. o f this enzyme ranges between 1 2 0 , 0 0 0 and 3 0 0 , 0 0 0 . Its p re fe r red template is poly A-oligo d T , it is st imulated by 2 0 0 m M KC1 and it is inhibited by N E M .

Since condit ional mutat ions in the genes specifying for the various D N A polymerase species a re not available in eukaryot ic cells, d ifferent approaches have been used to assign a role for each o f them: a) the levels o f D N A pol a have been found to increase in rapidly growing cells, such as those from regenera t ing rat liver ( 1 0 9 ) and neoplasic liver ( 1 1 0 ) , and also dur ing the S period o f the cell cycle ( 1 1 1 ) . No changes in the level of D N A pol 3 have been observed unde r these condit ions, a l though D N A pol 7 levels have also expe r imen ted a rise; b) the effect of the inhibitors 2 ' 3 ' d ideoxy T T P ( d d T T P ) and a r a C T P o n D N A r e p l i c a t i o n h a s b e e n examined. D N A synthesis in isolated S-phase H e L a nuclei ( 1 1 2 ) and in permeabi l ized baby hamste r kidney cells ( 1 1 3 ) is very sensitive to a r a C T P , while purified D N A pol B is resistant to this inhibitor. O n the o the r hand, d d T T P , which inhibits D N A pols 3 and 7 but not D N A pol a , does not inhibit e i ther in vitro S V 4 0

( 1 1 4 , 115) of H e L a cell ( 1 1 2 , 116) D N A synthesis; c) a third approach has been to identify the different D N A polymerase species present in replication complexes o f some animal viruses. Thus, D N A pols a and 7, but not DNA pol 3, are present in adenovirus replication complexes ( 1 1 7 - 1 1 8 ) , while D N A pol a is found in S V 4 0 replication complexes ( 1 1 4 , 115 , 119) . Addition of d d T T P to isolated nuclei which synthesize adenovirus D N A in vitro has a s t rong inhibitory effect, indicating that D N A pol 7 is required for viral r e p l i c a t i o n ( 1 2 0 ) ; d ) n u c l e i f r o m a d u l t non-dividing brain neurons contain only D N A pol 3 ( 1 2 1 ) . B y st imulating repair-type synthesis with U V light, a seven- to ten-fold stimulation o f D N A repair at tr ibutable to D N A pol B e a n be observed ( 1 2 1 , 122 ) . Also in these cells, evidence has been obtained for a role o f D N A pol 7 in mitochondria l D N A replication, since this is the only D N A polymerase species present in this organel le (106 , 123) ; e) Spadar i and Weissbach ( 1 2 4 ) have found that only D N A pol a can ex tend a natural R N A pr imer hybridized to a D N A template. Pr imers in vivo consist mainly o f short RNA transcripts ( 5 3 , 1 2 5 - 1 2 7 ) .

T h e conclusions emerg ing from these studies seem to indicate the following: D N A pol a is the replicating enzyme in the nuclei . D N A pol 3 is a repai r enzyme and D N A pol 7 replicates mitochondria l DNA. However , the possibility that D N A pol a and D N A pol 3 have minor roles in repai r and replication respectively, cannot be ruled out.

4. S E A L I N G O F D N A F R A G M E N T S

Cells have an enzyme capable of j o i n i n g single-stranded interrupt ions which arise in D N A dur ing the processes of replication, recombinat ion and repair . T h i s enzyme, called D N A ligase, is essential for viability o f the cell ( 1 2 8 ) . I t was first found en E. coli using as an assay the enzymatic conversion o f l inear lambda D N A conta in ing sticky ends to a covalently closed circular form ( 1 2 9 , 130) . D N A ligase plays an active role in discontinuous D N A replication, linking the short Okazaki f ragments to the growing ch romosome .

2 2 0 VICUÑA

4.1. E . C O L I U N A L I G A S E

T h e enzyme has been purified to

homogenei ty ; it is a single polypeptide with a

M.W. o f 7 5 , 0 0 0 (reviewed in ref. 131) . T h e

sealing react ion requires M g 2 + and also N A D

as a cofactor , to form a l igase-AMP

intermedia te complex . T h e adenyl g roup o f

the coenzyme binds to an e-amino g roup o f a

lysine residue o f the enzyme, with the release

o f N M N . In a second step, the A M P moiety is

t ransferred to the 5 ' phospha te terminus of

D N A , in a react ion that requires the free 3 '

hydroxyl end . Th i rd ly , a phosphodies ter

bond is fo rmed by a nucleophi l ic attack, o f the

3 ' hydroxyl t e rminus to the activated 5' end ,

with the concomi tan t release o f A M P . Each

step o f this react ion can be reversed by the

cor respond ing products . A m m o n i u m ions

activate the enzyme. E. coli D N A ligase can

j o i n adjacent D N A fragments and also 3 ' R N A

to 5 'DNA, only when they are hybridized to a

D N A strand.

4.2. BACTERIOPHAGE T 4 GENE 30 PROTEIN

Phage T 4 induces ( 1 3 2 ) , as does T 7 ( 1 3 3 ) , a

D N A ligase which is the product o f gene 3 0 .

T 4 D N A synthesis is impaired in gene 3 0

mutants , the re fo re the phage- induced ligase

canno t be replaced by the host enzyme ( 1 3 4 ,

135 ) . Al though the same three-step

mechan ism out l ined for the E. coli enzyme is

also followed by T 4 ligase, both enzymes

differ in several aspects: a) A T P is the cofactor

o f the phage enzyme, be ing A M P and

pyrophosphate the products o f the react ion

instead o f A M P and N M N ; b) T 4 D N A ligase

can pe r fo rm end-to-end j o i n i n g o f flush

ended D N A molecules , conver t ing ds phage

P22 D N A into ol igomers ( 1 3 6 ) ; c) in contrast

to the host enzyme, T 4 D N A ligase can j o i n

D N A o r R N A segments in all combinat ions ,

e i ther hybridized to D N A o r R N A ( 1 3 7 , 1 4 0 ) .

4.3. MAMMALIAN DNA LIGASES I AND II

T h e major D N A ligase activity present in

extracts p repared f rom mouse embryo

fibroblasts ( 1 4 1 ) , c a l f thymus ( 1 4 2 ) , rat liver

( 1 4 3 ) and a h u m a n cell l ine ( 1 4 4 ) is called

D N A ligase I . I t is an A T P - requi r ing enzyme

with a M.W. ranging from 1 7 5 , 0 0 0 to 2 0 0 , 0 0 0 .

T h e mechanism of the ligation reaction

appears to be similar to that observed with

D N A ligases o f prokaryot ic origin, since the

format ion o f l igase-AMP and D N A - A M P

intermediates has been detected ( 1 4 5 , 146) .

Mammal ian cells also contain a second

enzyme with an A T P - d e p e n d e n t D N A ligase

activity, called D N A ligase I I ( 1 4 2 ) . Im­

munological tests indicate that this enzyme

is not related to D N A ligase I ( 1 4 7 ) . Bo th

enzymes are present in the nuclei , a l though

only D N A ligase 1 levels appea r to be regulat­

ed dur ing the cell cycle ( 1 4 7 ) .

5. CONFORMATIONAL CHANGES OF DNA DURING REPLICATION

St rand separat ion at the replication fork, as

well as the el iminat ion o f the topological

constraints arising dur ing the overall process

o f duplication, a re problems inheren t to D N A

synthesis.

A m o n g the proteins that alter D N A

conformat ion are the ss D N A binding

proteins , also called hel ix destabilizing

proteins ( H D P ) . T h e y have been isolated from

a variety o f prokaryot ic and eukaryot ic

sources, and they are thought to play several

roles in the cell: a) HDPs are requi red for the

pr iming react ion o f the ss D N A phage-s and

most probably o f the E. coli c h r o m o s o m e ;

b) they activate the e longat ion reaction

catalyzed by D N A polymerases; c) HDPs

facilitate unwinding catalyzed by D N A

helicases by binding to ss D N A at the

replication fork.

Enzymes that al ter the deg ree o f

supercoiling o f D N A are called topo-

isomerases. A m o n g them, nicking-

closing enzymes (swivelases) r emove super-

helical turns f rom circular dup lex D N A

to yield a re laxed and covalently closed

molecule . T h e react ion involves the

introduct ion o f a t ransient nick into the helix,

which allows the strand to swivel in o rde r to

relieve the torque crea ted dur ing the

unwinding o f the duplex . As nicking-closing

enzymes opera te without an energy source , it

has been proposed that a covalent

enzyme-DNA complex conserves the energy

o f the phosphodies ter bond dur ing the

ENZYMOLOGY OF DNA REPLICA H O N 2 2 1

relaxat ion react ion. Swivelases lack D N A ligase-type activity. T h e i r part icipation in D N A replication is only assumed, since no mutans have been isolated to date. O n the contrary, it has been clearly established that E. coli D N A gyrase, an enzyme that introduces superhel ical turns into D N A in an A T P - d e p e n d e n t react ion, plays an active role in D N A synthesis (as well as in t ranscript ion and recombina t ion) . T h e r e is now good evidence showing that D N A must be supertwisted in o r d e r to be replicated ( 3 1 , 1 4 8 - 1 5 1 ) . In addition, D N A gyrase is the target enzyme o f nalidixic acid and novobiocin, two potent inhibitors o f D N A synthesis ( 1 5 2 ) .

Topoisomerases can be assayed by agarose-gel e lectrophoresis , where supercoiled circular duplex D N A can be separated from re laxed D N A . ( 1 5 3 ) . S ince the reaction catalyzed by both nicking-closing enzymes and D N A gyrase does not follow a single hit mechanism, the separat ion of topoisomers with an in termedia te degree o f superhelici ty can be achieved.

Actual strand separat ion is carr ied out by DNA helicases. T h e s e enzymes catalyze the hydrolysis o f A T P to A D P and Pi in a reaction coupled to strand separat ion. S o m e o f them, like £ . coli rep protein and the r e c B C nuclease, require in addition the presence o f a H D P to avoid reassociation o f the D N A strands. T h e substrate utilized to measure the activity of DNA helicases consist in a labelled polynucleotide (e.g. a restrict ion f ragment ) hybridized to non-labelled ss D N A . T h e degree o f unwinding can be quant i ta ted as the amount o f radioactive D N A which is r endered acid soluble after t rea tment o f the product with a ss specific deoxyr ibonuclease . Helicases have been isolated from E. coli, bacter io-phage-infected cells and mammal ian cells.

5.1. E. COLI HDP

E. coli D N A binding protein is a heat stable te t ramer o f 1 8 , 5 0 0 M.W. subunits ( 1 5 4 ) which binds cooperatively to ss D N A . In the absence o f M g 2 + and at low ionic s trength, it lowers the melting tempera ture o f the duplex by nearly 40°C ( 1 5 5 ) . Each t e t r amer o f the native protein interacts with 32 nucleot ides, causing

a 4 0 percent shor tening in the length of ss DNA ( 1 5 5 ) . B ind ing o f E. coli H D P to D N A reduces the activity o f most ss DNases , excep t micrococcal nuclease, E . oy/2 exonuc lease I and the nucleases associated with E. coli D N A pol II and T7- induced D N A pol ( 1 5 6 ) . Mol ineux el al. ( 156 ) have shown that, excep t for micococcal nuclease, these enzymes form stable complexes with the binding protein. Moreover , the D N A pol I I - H D P c o m p l e x is able to copy a long gap in dup lex D N A , a reaction the free polymerase cannot carry out (157) .

In vitro, E. coli H D P is required for the convers ion of ss D N A o f icosahedral and f i lamentous bacter iophages to the ds replicative form. (3) . Its role in D N A replication in vivo became evident only recently, when Meyer et al. ( 1 5 8 ) identified a mutant with a temperature-sensi t ive defect in E. coli H D P .

5.2. BACTERIOPHAGE-CODED HDPs

— T4 gene 32 protein

T h e product o f T 4 gene 32 (M.W. 3 5 , 0 0 0 ) was the first H D P isolated ( 1 5 9 ) . W h e n gene 32 protein binds to ss D N A at saturat ing levels.it covers about eight nucleotides, ex tend ing the D N A chain 5 0 percent ( 1 6 0 , 1 6 1 ) . In this ss DNA-pro te in complex , T 4 H D P forms l inear aggregates in which the D N A binding sites a re thought to be al igned. T h e s e protein-protein interact ions could be the basis for cooperat ive binding to D N A . G e n e 32 protein stimulates specifically T 4 D N A pol when assayed with a long ss D N A template ( 1 0 1 ) and also with nicked ds D N A ( 1 6 2 ) ; in the later case, it does it presumably by allowing the d isplacement o f the non- templa te s trand. T 4 H D P plays a p rominen t role in D N A metabol ism, since it is requi red for replication ( 1 6 3 ) , repai r ( 1 6 4 ) , and genet ic recombina t ion ( 1 6 5 ) .

— T7 HDP

T 7 D N A binding protein (M.W. 3 0 , 0 0 0 ) stimulates specifically T 7 D N A pol ( 1 6 6 , 167 ) . I t has not been def ined genetically, possibly because it can be replaced by E. coli H D P . T 7 HDP lowers the T m o f poly d A T by 4 0 ° C , and

222 VICl'ÑA

aggregates itself in the p re sen te of M g 2 +

( 1 6 8 ) .

— fd gene V protein

Bac te r iophage fd-coded binding protein is

absolutely required to direct the synthesis of

progeny ss viral D N A ( 1 6 9 , 170) . The native

protein consists of a d imer of 2 0 , 0 0 0 , each

m o n o m e r covering approximate ly four nu­

cleotides ( 1 7 0 , 171) . Coopera t ive binding is so

strong, that fd H D P lowers the I'm o f ds DNA

by nearly 40°C ( 1 6 9 ) . The t e r c i a n s tructure o f

gene V product o f bac te r iophage fd and its

complexes with D N A have been studied ( 1 7 2 ) .

T h e enzyme binds to c i rcular single strands in

such a way that brings toge ther non adjacent

regions o f the D N A , giving to the complex a

rod-like shape ( 1 7 3 ) . .,

5.3. EUKARYOTIC HDPs

Proteins have been isolated from plant ( 1 7 4 ) ,

mammal ian ( 1 7 5 - 1 8 0 ) and viral infected cells

( 1 8 1 , 182) by virtue o f their preferent ia l bin­

ding to ss D N A . T h i s property, c o m m o n to

their prokaryot ic counterpar ts , besides facili­

tating mel t ing o f a duplex, promotes also re-

naturat ion o f single strands, a react ion requi­

red dur ing recombina t ion and repair . HDPs

isolated from the various eukaryot ic sources

activate specifically homologous D N A poly­

merases ( 1 0 8 , 178 , 183) , indicating a possible

involvement o f these proteins in the replicati-

ve process in vivo.

Nuclei isolated from meiotic cells o f lilies

and some mammals conta in an H D P (M.W.

3 2 , 0 0 0 ) which is only active dur ing the inter­

val following the S-phase until te rminat ion o f

c h r o m o s o m e pair ing ( 1 7 4 , 184 ) . T h i s enzyme,

as well as H D P isolated from mouse ascites

cells, changes its binding capacity to ss and

ds DNA upon phosphorylat ion; which may

constitute a regulatory mechanism ( 1 7 8 ,

185) .

H u m a n adenovirus induces an H D P (its

M . W . varies with the type o f virus) which has

several functions: a) it is involved in the initia­

tion and e longat ion steps o f viral D N A repli­

cation ( 1 8 6 , 1 8 7 ) ; b) it modulates the levels o f

several early viral t ranscripts , including auto-

r e g u l a t i o n o f i ts o w n leve l s ( 1 8 8 , 1 8 9 ) ;

c) t empera tu re - sens i t i ve mutan t s in a d e n o

H D P transform rat cells at non permissive

t empera tu re at h igher frequencies than wild

type virus ( 1 9 0 ) ; d) the enzyme is also requi­

red for in vitro viral replication. When this

reaction contains endogenous viral DNA as

template , adeno H D P can be replaced by E. coli H D P ( 1 9 1 , 192) . Adeno- induced H D P is

present as a phosphoprote in that exhibits dif­

ferent degrees of phosphorylat ion ( 1 9 2 - 1 9 5 ) .

T h i s modificat ion, however, does not affect

the binding of this protein to D N A .

5.4. BACTERIAL DNA I OPOISOMERASES

—Swivelases

E. coli omega protein was the first

nicking-closing enzvme to be discovered

(196) . Since then, they have been isolated

from M. luteus ( 1 9 7 - 1 9 8 ) , Pseudomonas B A L - 3 1 ( 1 9 9 ) and .4. tumejaaens ( 2 0 0 ) . B a c ­

terial swivelases (M.W. about 1 1 0 , 0 0 0 ) art-

designated type I topoisomerases . since they

tan readily remove negative superhelical

turns but remove positive turns inefficiently.

T h e y all require only Mg~ + ion for activity,

except the enzyme from Pseudomonas B AL-3 1,

which requires in addit ion a monovalent

eation salt.

In the absence of M g 2 + , E. coli omega

protein forms stable complexes with ss DNA,

which dissociate when minute amounts of

M g 2 + are added ( 2 0 1 ) . T r e a t m e n t o f the

complex with alkali o r pronase leads to the

eleavage of the D N A chain, after which the

protein remains covalently l inked to the 5 '

terminus o f the DNA.

It has been demons t ra ted that the ss

character o f a duplex D N A molecule is

proportional to its deg ree of negative

supercoiling ( 2 0 2 ) . T h e r e f o r e , the removal o f

only negative superhelical turns, the

dependency on the degree o f supercoi l ing for

catalysis and the inhibition of the reaction by

ss DNA, are consistent with the initial binding

of bacterial swivelases to ss regions o f the

substrate in o r d e r to catalyze the relaxation

reaction.

E. coli nicking-closing enzyme also forms

salt stable complexes with ds D N A , al though

they are nei ther dissociated by the addit ion o f

M g 2 + , nor does D N A scission occur when

protein denaturants are added ( 2 0 3 ) .

E N Z Y M O L O G Y O K U N A R E P L I C A T I O N 2 2 3

Two novel react ions a re catalyzed by the E. coli and M. luteus enzymes with c i rcular ss D N A : a) in the presence of monovalent cations they form knot ted rings which can be identified by sedimentat ion and e lect ron microscopy ( 1 9 7 , 2 0 4 ) , and b) they p romote the interwinning o f circles of complemen ta ry sequences into a covalently closed duplex r ing ( 2 0 5 ) .

— DNA gyrase

D N A gyrase is an enzyme that induces negative supercoi l ing into closed ci rcular ds D N A at the expense o f A T P hydrolysis ( 2 0 6 ) . It has been isolated from E. coli ( 2 0 6 ) and M. luteus ( 2 0 7 ) , a l though most o f the studies have been pe r fo rmed with the fo rmer enzyme. D N A gyrase is an essential enzyme in E. coli ( 2 0 8 ) and it is involved in: a) replication o f 0 X 1 7 4 ( 1 4 8 ) , some plasmids ( 3 1 , 2 0 9 ) , T 7 ( 2 0 1 , 2 1 1 ) and host D N A s ( 1 4 9 ) ; b) integrative recombina t ion o f phage lambda ( 2 1 2 ) ; c) D N A repa i r ( 2 1 3 ) and, d) t ranscript ion ( 2 1 4 - 2 1 8 ) . Repl icat ion o f phage T 4 mutants in DNA-delay genes 3 9 , 52 and 6 0 unde r non permissive condit ions, also requires E. coli D N A gyrase ( 2 1 9 ) . T h e s e genes code for the componen t s o f a phage A T P - d e p e n d e n t topoisomerase (see below). In addit ion to A T P , gyrase requires M g 2 + and it is activated by spermidine ( 2 0 6 ) .

T h e purif ied enzyme can catalyze five dif ferent react ions: a) the int roduct ion o f negative superhelical turns in the presence o f A T P ; b) re laxat ion o f supercoils in the absence o f A T P ; c) D N A - d e p e n d e n t hydrolysis o f A T P to A T P and Pi; d) enzyme binding to D N A , e) site-specific cleavage o f D N A .

D N A gyrase is composed o f two subunits, A and B , which can be purif ied separately and r ecombined to reconst i tute active D N A gyrase ( 2 2 0 - 2 2 2 ) . Apparent ly , both subunits a re present independent ly in the cell, as well as forming a complex . Subuni t A is coded by nal A gene , which governs sensitivity to nalixidic and oxol inic acids ( 2 1 8 , 2 2 1 , 2 2 0 , 2 2 3 ) . W h e n purif ied separately to homogenei ty , nal A gene product is a d imer o f two identical subunits, each with a M.W. o f 1 1 0 , 0 0 0 . Nal A gene product catalyzes, as does native D N A

gyrase, the A T P - i n d e p e n d e n t and nalidixic acid sensitive relaxat ion o f positive and negative superhel ical turns o f D N A , there fore it is a type II topoisomerase ( 2 2 0 , 2 2 3 ) . Several cr i ter ia indicate that in spite o f their similar M . W . and activities, the nal A protein is not related to the o m e g a protein ( 2 2 0 ) . Subuni t B o f D N A gyrase is coded by thecou g e n e , which governs sensitivity to coumermic in and novobiocin ( 2 2 4 ) . Gel ler t et al. have found that the c o u , gene product (M.W. 9 5 , 0 0 0 ) is involved in the energy t ransduct ion aspect o f the supercoi l ing react ion and that this protein has a specific binding site for A T P which is b locked by novobiocin ( 2 2 5 - 2 2 7 ) . Native D N A gyrase is able to induce negative superhel ical turns in the p resence o f nonhydrolyzable A T P analogs ( 2 2 6 ) . T h e r e f o r e , Cozzarelli et al. have proposed that A T P acts as an allosteric ef fec tor which is hydrolyzed to A D P and Pi in o r d e r to make the enzyme work catalically ( 2 2 6 ) .

W h e n oxol inic acid and S D S are added to the react ion mix ture , D N A gyrase cleaves ds D N A site-specifically ( 2 2 0 , 2 2 3 , 2 2 5 , 2 2 6 , 2 2 8 ) . I t makes a four nucleot ide s taggered cut, c reat ing D N A termini with a 3 ' hydroxyl end and a 5 ' ex tens ion which contains the enzyme covalently bonded ( 2 2 8 ) . D N A gyrase follows then the pat tern o f type I and type I I topoisomerases , storing the energy released by scission o f the D N A backbone in a covalent enzyme-DNA intermedia te .

5.5 BACTERIOPHAGE-CODED DNA

TOPOISOMERASES

— T4 DNA topoisomerase

A distinct A T P - d e p e n d e n t D N A topo­isomerase has been isolated from T 4 infected cells ( 2 2 9 , 2 3 0 ) . T h e purified en­zyme preparat ion exhibits th ree protein componen t s : the product o f the DNA-delay gene 3 9 (M.W. 6 4 , 0 0 0 ) , the product o f the DNA-delay gene 5 2 (M.W. 5 1 , 0 0 0 ) and a third protein (M.W. 1 1 0 , 0 0 0 ) the identify o f which is not known. T 4 topoisomerase hydrolyzes A T P during the relaxat ion o f both positive and negative superhelical turns. T h e enzyme is not inhibited by the antibiotics oxol inic acid and novobiocin, which are known antagonists

2 2 4 V I C U Ñ A

oí E. coli D N A gyrase. T 4 D N A topoisomerase

is not essential for viral replication, since it can

be replaced by E. coli D N A gyrase ( 2 1 9 ) .

Under these condit ions, however, phage

DNA synthesis is depressed.

Liu et al. ( 230 ) have proposed that the

enzyme may act at the origin site in conjunc­

tion with the products o f phage genes 41 and

61 (pr imase) in o r d e r to initiate D N A synthe­

sis. I t remains to be seen i f this A TP-

dependen t T 4 topoisomerase is able to induce

superhel ical turns on ds D N A , as well as to

establish the role o f A T P in the relaxat ion

react ion.

—Lambda int gene product

This phage-coded enzyme, which is required

for integrative recombinat ion , contains

swivelase activity ( 2 3 1 ) . T h e int gene product

is a type II topoisomerase , since it re laxes

positive and negative supercoils . T h e

nicking-closing activity o f int protein shows

no sequence specificity and functions in the

absence o f a divalent cation. It is inhibited by

M g 2 + ions, spermidine and ss D N A .

—0X174 gene A and fd gene II proteins

T h e s e enzymes, which are site-specific

endonucleases requi red to start replication o f

the ds replicative forms 0 X 1 7 4 and fd

phages , ( 5 9 , 6 7 ) also display nicking-closing

activity.

W h e n 0 X R F I D N A is incubated with low

levels o f A protein, a small p ropor t ion o f

re laxed D N A is detected ( 6 0 ) . T r e a t m e n t o f

re laxed 0 X R F I V D N A with sarkosyl o r S D S

has no effect in its s t ructure. I n contrast ,

t reatment with phenol and/or prote inase K

quantitatively yields nicked R F I I D N A ( 6 0 ) .

Incubat ion o f 0 X R F I with large amounts o f A

protein yields R F I I with large detectable

re laxed structures.

U p o n t rea tment o f fd supercoi led D N A

with gene I I endonuclease , two thirds o f this

substrate are cover ted to the nicked form and one third is t r ans formed to re laxed covalently closed circles. T h i s addit ional activity o f gene I I protein might be impor tan t for the circularization o f the l inear viral s trands dur ing phage D N A synthesis ( 6 8 ) .

5.6 EUKARYOT1C DNA TOPOISOMERASES

T h e eukaryot ic enzymes have been te rmed

type I I topoisomerases since they can catalyze

relaxat ion o f both positive and negative su­

perhelical turns. They have been found in

mouse ( 2 3 2 ) , drosophi la eggs ( 2 3 3 ) , K B cells

( 2 3 4 ) , H e L a cells ( 2 3 5 ) , rat iiver nuclei ( 2 3 6 ) ,

vaccinia virions ( 2 3 7 ) , yeast ( 2 3 8 ) , e tc . In addi­

tion to their possible role at the replication

fork, they part icipate in the association of his­

tories to the c h r o m o s o m e s ( 2 3 9 ) . Eukaryot ic

swivelases exhibi t M.W. ranging from 6 0 . 0 0 0

to 8 0 . 0 0 0 and posses several features that dis­

tinguish t hem from swivelases o f bacterial ori­

gin: a) they requi re between 0 .15 M and 0 . 2 0

M monovalen t cation salt; b) they can be as­

sayed in the p resence o f excess E D T A , and c)

their activity does not d e p e n d on the deg ree

o f superhelici ty o f the substrate. O n the o the r

hand, like type I topoisomerases , they do not

requi re an energy source and catalyze the

stepwise relaxat ion of the supercoi led subs­

trate.

Work ing with the rat liver enzyme,

C h a m p o u x ( 2 4 0 - 2 4 2 ) has been able to t rap a

nicked p ro te in -DNA in te rmedia te c o m p l e x

which contains the enzyme covalentfy

a t tached to the 3 ' end . T h e chemica l na ture o f

this l inkage is not known. With respect to

specificity, the enzyme can act at any site on ds

DNA.^However , studies carr ied out with the

S V 4 0 c h r o m o s o m e , show that rat liver

nicking-closing enzyme has a p re fe rence for

six discrete sites ( 2 4 3 ) . C h a m p o u x et al. have

also demons t ra ted that in contras t to o the r

enzymes involved in D N A replicat ion, rat liver

topoisomerase levels a re not regula ted dur ing

the cell cycle ( 2 4 3 ) .

Recent ly , a D N A topoisomerase has been

purif ied f rom rat liver mi tochondr ia ( 2 4 4 ) .

T h e enzyme sensitivity to e thidium b romide

and to the t rypanocidal d rug bereni l is

d i f ferent to that o f its nuc lear counte rpar t ,

suggesting that they may be dif ferent en­

zymes.

5.7. E. COLI DNA HELICASES

— DNA helicase I

E. coli helicase I is a long, f ibrous single

polypeptide (M.W. 1 8 0 , 0 0 0 ) which is present

KN/.VM()l.(X;V ()!• 1)NA RK.I'l.ICAHON 2 2 5

at about 6 0 0 copies per cell ( 2 4 5 - 2 4 7 ) . The enzyme has a ss DNA-dependen t N P T a s e activity: A T P and d A T P are the pre fe r red substrates, a l though the o the r r N T P s are hydrolyzed to some exten t . T o initiate unwinding o f ds DN A, helicase I binds first to pro t ruding 5' ss ends which have to be at least 2 0 0 nucleotides long. It then advances in the 5' to 3 ' direct ion o f the D N A chain to which it is bound , in a react ion coupled to A T P hydrolysis. A D N A molecule with prot ruding 3 ' ss ends is not a substrate for helicase 1 ( 2 4 8 , 2 4 9 ) . Abou t 7 0 molecules o f enzyme are present continously at the separat ion fork. Since the protein has a s t rong tendency to ' form aggregates in solution, this type o f protein-protein interact ion may be the basis for cooperativity and translocation o f the enzyme at the replication fork.

A T P a s e activity o f helicase I is opt imal under condit ions where the enzyme is known to form aggregates and it is abolished when ds DNA is used as a cofactor .

D N A h e l i c a s e I w o r k s in a p r o c e s s i v e fashion: i f ss D N A is added once the react ion has started, there is no inhibit ion o f the unwinding react ion ( 2 4 8 , 2 4 9 ) .

— DNA helicase II

E. coli helicase IITM.W. 7 5 , 0 0 0 ) ( 2 5 0 , 2 5 1 ) is probably identical to D N A - d e p e n d e n t A T P a s e I isolated by Kohiyama's g roup ( 2 5 2 , 2 5 3 ) . T h e enzyme shows specificity for A T P or d A T P hydrolysis and it is inactive with o the r nucleotides ( 2 5 0 ) . As helicase I , it starts unwinding ds D N A sequentially in the 5 ' to 3 ' direct ion, a l though it requires ss ends only 12 nucleot ides long ( 2 4 8 , 2 4 9 ) . In contrast to helicase I , helicase.11 requires the cont inous absort ion o f enzyme molecules to D N A at the separation fork. T h i s s toichiometr ic require­ment o f enzyme is consistent with the inhibition observed when compet ing ss D N A is added to the assay.

— rep protein

A third E. coli D N A helicase is coded by the rep gene . T h i s protein (M.W. 7 0 , 0 0 0 ) is requ i red for the replicat ion both in vito and in

vitro of certain bac te r iophage genomes ( 0 X 1 7 4 , fd, P2) and for the ' normal replication o f the host c h r o m o s o m e ( 6 3 , 2 5 4 - 2 5 7 ) . T h i s r equ i r emen t has provided an assay for the purification o f r ep protein and the subsequent study o f its physiological role ( 6 2 - 6 4 , 2 5 8 - 2 6 1 ) .

Replicat ion in vitro o f ds 0 X 1 7 4 R F I requires the phage coded A protein, rep protein, E. coli H D P and D N A pol I I I plus e longat ion factors (62 , 6 4 ) . First, i the A protein cleaves site-specifically the viral s t rand o f 0 X 1 7 4 R F I and remains covalently bound to the 5 ' end o f the nick ( 6 0 ) . T h e dup lex D N A is then unwound by the rep protein in the presence o f H D P with the s imultaneous hydrolysis o f two A T P (or d A T P ) molecules per base pair b roken ( 2 5 8 - 2 6 2 ) . Nicked 0 X 1 7 4 R F lacking A protein is not a substrate no r a cofac tor for rep A T P a s e , suggesting that the A protein present at the nick interacts with rep to start unwinding (62 , 2 6 1 ) . In the absence o f A protein, but in the presence o f HDP, rep enzyme unwinds ds D N A when it has ss extending regions. As D N A helicases I and I I , rep protein unwinds in the 5 ' to 3 ' direc­tion ( 2 6 1 ) . T h i s is impor tan t i n ' t he react ion descr ibed above, because the 3 ' end o f the A prote in- induced nick is left in a duplex structure, to be used as a p r imer for viral replication via rolling circle mechanism.

A T P a s e activity o f rep protein requires ss D N A as a cofactor ; ds D N A o r HDP-coa ted ss D N A are inactive as cofactors . However , since the rep catalyzed unwinding react ion requires HDP, it appears that the t rue ef fec tor for the A T P a s e is the separat ing fork ( 2 6 1 , 2 6 2 ) .

— rec BC nuclease

E. coli rec B C nuclease, also called exonuclease V , plays a centra l role in genet ic recombina t ion and recombinat ional repai r ( 2 6 3 ) . T h e native enzyme is a composi te protein o f M.W. 2 7 0 , 0 0 0 which exhibits several activities: a) it is an A T P - d e p e n d e n t 5 ' to 3' and 3' to 5' exonuclease , which degrades both ds and ss D N A ; b) it is also an A TP-stimulated ss endonuc lease and c) it is a D N A dependent A T P a s e , a l though s imultaneous DNA degradat ion is not necessary for A T P hydrolysis ( 2 6 4 , 2 6 5 ) .

2 2 6 VICUÑA

S. L inn has proposed a model for the

degradat ion o f ds D N A by re t B C nuclease

( 2 6 6 ) : the enzyme binds to both strands at the

end o f the duplex. It then unwinds the strands

from o n e t e rminus , cleaving f ragments at 5 0 0

nucleot ide intervals while it remains bound to

the end o f the undegraded strand. Af ter

unwinding about 5 , 0 0 0 nucleotides, the

enzyme switches strands and degrades the ss

tail f rom its terminus , yielding a shor tened

wholly duplex molecule . T h e 5 0 0 nucleotides

long f ragments a re then degraded by the

enzyme to acid soluble ol igonucleot ides.

Presumably, A T P hydrolysis is coupled to the

unwinding o f the D N A helix and it is not

related to the actual nucleolytic activity, since

several A T P molecules a re hydrolyzed per

each phosphodies ter bond broken ( 2 6 4 , 2 6 7 ) .

I f H D P is added to this react ion, rec B C

works as a D N A helicase ( 2 6 8 , 2 6 9 ) . B ind ing

o f H D P to the ex tend ing ss tails results in

protect ion against exonucleolyt ic

degradat ion. T h e r e f o r e , the products a re

long ss pieces derived from limited nuclease

cutt ing after helix unwinding.

R e c B C enzyme is regulated by C a 2 + ions.

W h e n both C a 2 + and M g 2 + are present

simultaneously in the react ion, a marked

inhibit ion o f the nuclease activity is observed,

while the A T P a s e is not affected ( 2 6 9 , 2 7 0 ) .

U n d e r these condit ions, the enzyme binds

initially to the te rminus o f duplex D N A and

then tracks a long the D N A molecule ,

producing local and transient unwinding o f

the strads as it moves. As a result, ds D N A

remains as a dup lex s tructure, in spite o f the

fact that it contains a few n i c k s . T h e c o m b i n e d

action o f M g 2 + , C a 2 + and H D P produces

complete denatura t ion o f ds D N A and solely

ss f ragments a re found ( 2 6 9 , 2 7 0 ) .

5.8. BACTERIOPHAGE-CODED DNA HELICASES

— T4 dda protein

The purified product o f 1*4 gene dda is

DNA-dependent A T P a s e (M.W. 5 6 , 0 0 0 )

which is able to unwind ds D N A in the 5' to 3 '

direction ( 2 4 8 , 2 4 9 , 2 7 1 ) . The enzyme action

is non processive as indicated by the following

criteria: a) the n u m b e r of enzyme molecules

required to unwind D N A is a function o f the

lenght o f the DNA; b) the reaction is inhibited

when ss DNA is added to t rap Iree en/.yme

molecules. As most DNA helicases, T 4 - d d a

protein binds first to a ss region and (ba in

separation is brought about by the cont inued

adsortion o f protein molecules to DNA at the

separating fork. T h i s reaction is driven bv

A T P (or d A T P ) hydrolysis. Mutational loss o f

this enzyme is not lethal to the virus ( 2 7 2 ) .

— T7 gene 4 protein

T h i s enzyme does not only fuction as a D N A

primase for T 7 replication ( 4 5 - 4 8 ) , but also as

a nucleoside t r iphosphate-dependent D N A

unwinding catalyst ( 4 5 , 4 9 , 2 7 3 - 2 7 5 ) . T 7 D N A

pol is unable to use intact o r nicked duplex

D N A as template . However , in the presence o f

gene 4 protein and the four d N T P s , T 7 D N A

pol catalyzes the incorporat ion of d N T P s ,

which is started at a nick in ds D N A (49 ,

2 7 3 - 2 7 5 ) . T h i s activation o f the polymerase by

gene 4 protein is specific. Polymerization

occurs simultaneously with the displacement

o f o n e of the parental strands, with the

concomi tan t hydrolysis o f 4 .6 d N T P s to

dNDPs and Pi for each d N T P polymerized

( 2 7 3 ) . At later t imes in the react ion, the

polymerase j u m p s across the fork and copies

the displaced strand, the re fo re all the newly

synthesized D N A is covalently a t tached to the

template ( 2 7 4 , 2 7 5 ) .

I f the react ion mix tu re with nicked D N A

contains both d N T P s and r N T P s , 17 gene

4 unwinding protein synthesizes oligo-

ribonucleotides on the displaced ss tail which

function as pr imers for T 7 D N A pol. As a

result, D N A chains copied in the lagging

strand are not covalently at tached to the D N A

template (47 , 4 8 , 2 7 4 ) . U n d e r these

condit ions, the total n u m b e r o f d N T P s and

r N T P s hydrolyzed per d N T P incorpora ted

are 3 .0 and 1.2, respectively ( 2 7 3 ) . B o t h

p r imer and D N A synthesis a re st imulated

about 5-fold by e i ther E. coli o r T 7 binding

proteins ( 4 7 ) .

T 7 gene 4 protein is a ss DNA-dependen t

r N T P a s e or d N T P a s e ( 4 5 , 2 7 3 ) . Dup lex D N A

is a cofac tor only when hydrolysis o f

nucleoside t r iphosphates is coupled to D N A

synthesis; d d T T P , an inhibi tor o f T 7 D N A

pol, stops N T P a s e activity. Moreover , 3 — 7

E N Z Y M O L O G Y O F D N A R E P L I C A T I O N 2 2 7

methylene d T T P , which cannot be

hydrolyzed to T D P and Pi, inhibits also D N A

synthesis ( 2 7 3 ) .

5.9. EUK.ARYOTIC DNA HELICASES

Cobianchi et al. ( 2 7 6 ) have isolated from

human cells a ss D N A - d e p e n d e n t A T P a s e

(M.W. 1 1 0 , 0 0 0 ) . T h e enzyme binds first to ss

D N A and moves a long in the 5 ' to 3 ' direct ion

unwinding dup lex D N A . H u m a n A T P a s e can

activate D N A pol 6 from H e L a cells possibly

by providing it with a template upon

denatura t ion o f the duplex . d N T P

incorporat ion is e n h a n c e d even futher by ca l f

thymus HDP.

In contras t to E. coli helicase I, human

helicase is unable to unwind R N A fragments

hybridized to D N A .

6. CONCLUSIONS

A b r i e f outl ine o f some o f the proteins

involved in D N A replication has been

presented. As stated before , the re is direct

p r o o f for the part icipation o f most o f them in

this process. O t h e r proteins , like lambda int

gene product and rec B C nuclease a re not

involved in D N A synthesis, but they have been

included due to their similarity with

replicat ing proteins .

Al though all these enzymes have been

discussed independent ly , o n e has to imagine

all o f them working as a repl isome complex at

the replication fork.

D N A synthesis starts at a specific origin and

proceeds almost continously in the leading

strand. At t h e same t ime, helicases with the

help o f H D P s unwind the duplex , and

discont inuous synthesis proceeds in the

lagging strand. Pr iming o f Okazaki f ragments

may requi re several proteins and elongat ion

proteins. La te r , Okazaki f ragments a re

processed and sealed by D N A ligase to form a

cont inuous polynucleot ide chain.

S o m e aspects o f D N A synthesis remain still

obscure . Not much is known about its

regulat ion and the mechan i sm o f te rminat ion

o f c h r o m o s o m e replication. T h e answer o f

these quest ions presents cer tain difficulties,

since the various systems which have been

investigated, a l though shar ing a c o m m o n

genera l pat tern, present also several different

features. F o r example , a c i rcular duplex

molecule may replicate th rough a rolling

circle o r a Cairns ( tetha) type o f in termediate ,

or both. W e still have to learn the role o f

adenovirus terminal protein, the lambda

genes O and P and the T antigen of S V 4 0 in

the initiation react ion, e tc . The picture

complicates even fur ther when cons ider ing

the involvement o f enzymes that display more

than a single catalytic activity, such as D N A

gyrase, 0 X 1 7 4 A protein, fd gene II protein

and T 7 gene 4 protein.

Finally, it is interest ing to point out how

actively A T P participates in D N A replicat ion:

a) pr imases pre fe r to start with A T P at the 5 '

end ; b) A T P is required for the format ion of

specific complexes a m o n g proteins , i.e. dna

B-dna C, pr imed- template-e longat ion factor

I, etc.; c) A F P es a cofac tor for D N A ligases

and d) A T P is hydrolyzed by helicases, gyrase,

dna B , replication factor Y , T 4 coded

topoisomerase, T 4 gene 41 protein, e tc .

ACKNOWLEDGEMENTS

I am grateful to E. Cardemil, J . Eyzaguirre and A. Yudelevich for their help in the preparation of this manuscript.

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8. APPENDIX

The following translations into Spanish for the

names of the enzymes discussed in this paper are

proposed:

primase: enzima partidora.

DNA binding protein: proteína DNA-ligante.

HDP: proteína hélice-inestabilizadora. nicking-closing enzyme: enzima de corte y sellado. gyrase: girasa. helicase: helicasa.

swivelase: enzima DNA-relajante.