DNA Replication

DNA Replication

The basic rules for DNA replication

DNA synthesis at the replication fork

Termination of replication

Other modes of DNA replication

DNA Polymerases

Initiation of replication

Regulation of re-initiation


Circular bacterial chromosomes

Linear chromosomes

Replication fork

Replication fork

For the circular bacterial replicon, the two replication forks move around the genome to a meeting point.

Forks meet

E. coli

4,639,221 bp

K-12

Terminus

ori C

圖引用自： Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-1

Binding of Tus protein to a ter site arrests replication fork advancement.

E, D, A

ter sites:

C, B

Figure 13.7

Tus proteinterminator utilization substance

Encoded by the tus gene

A 309-residue monomer

圖引用自： Voet, D., Voet, J. G. and Pratt, C.W. (1999) Fundamentals of Biochemistry. John Wiley & Sons, Inc. Fig. 24-16

Figure 14.34

Tus binds to ter asymmetrically and blocks replication in only one direction.

Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-17b

Type II topoisomerases are required to separate daughter DNA molecules.

Nelson, D. L. and Cox, M. M. (2005) Lehninger Principles of Biochemistry. 4th Ed., Worth Publishers. Fig. 25-17b

(Type II topoisomerase)

Catenanes

The ends of linear DNA are a problem for replication.

3’5’

3’

5’3’

5’3’5’

3’5’

3’5’

3’5’

3’5’

Primer removal and ligation of Okazaki fragments

Last Okazaki fragment

+

Linear bacterial chromosome

3’5’

3’5’

3’5’

3’5’

+

+3’5’3’

5’

3’5’3’

5’

3’5’3’

5’

3’5’3’

5’

+

+

Replicate again

The chromosome becomes shorter.

Replication

forkReplication fork

Linear eukaryotic chromosome

DNA polymerase cannot synthesize the

extreme 5’ ends of linear DNA.

Lagging strand Leading strand

Leading strand Lagging strand

5’

5’3’

3’

3’ 5’

5’ 3’

Replication fork

Replication fork

How do cells solve the end replication problem?

Using terminally attached protein to provide an OH

Using telomerase to extend the ends of chromosome

(in certain species of bacteria)

(in eukaryotic cells)

Telomeres:

CentromereTelomere Telomere

the ends of eukaryotic chromosomes

(Tx Gy)n

(Ax Cy)n

Tx Gy Tx Gy

Telomeric DNA:

x, y : 1 ~4

n: 20 ~ 100 in single- cell eukaryotes;

> 1500 in mammals

3’

Human TTAGGG

Arabidopsis thaliana

TTTAGGG

Tetrahymena thermophila

TTGGGG

Saccharomyces cerevisiae

T(G)2-3(TG)1-6

Mammalian telomeres end in a large duplex loop.

T loop

取材自： Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., and de Lange, T. (1999) Mammalian telomeres end in a large duplex loop. Cell, 97: 503-514, Fig. 3.

The 3’ single-stranded end of the telomere displaces the homo-logous repeats from duplex DNA to form a t-loop. The reaction is catalyzed by TRF2.

Fig

ure

19

.30

Telomerase: a ribonucleoproteins

Telomeric DNA is synthesized and maintained by telomerase.

RNA: as a template

Protein: reverse transcriptase

Telomerase uses its RNA component to anneal to the 3’ end of ssDNA region of the telomere.

Telomerase moves to the newly synthesized 3’end.

RNA template directs addition of nucleotides to 3’ end of DNA

1 2

3 Figure 19.31

extended by telomerase

DNA synthesized by DNA Pol

RNA primer

5’ 3’

3’ 5’

5’

3’

3’

5’

5’

3’

3’

5’

Termination of replication Summary

The two replication forks of E. coli chromosome initiate at oriC, move around the genome and then meet at a ter site.

Binding of Tus protein to a ter site arrests replication fork advancement.

Type II topoisomerases are required to separate catenated daughter DNA molecules.

1. For circular bacterial chromosome:

Termination of replication Summary

Telomerase solves the end problem by extending the 3’ end of the chromosome. Telomerase is a reverse transcriptase and specifically elongates the 3’OH of particular ssDNA sequences using its own RNA as a template. Type II topoisomerases are also critical to the segregation of large linear daughter chromosomes.

2. For eukaryotic linear chromosome:

DNA Replication





DNA Polymerases



In bacterial cells, methylation at the origin may regulate initiation.

(Hemimethylated)

(Fully methylated)

Me

Figure 14.36

oriC contains 11 GATC repeats that are methylated on adenine on both strands.

Replication generates hemimethylated DNA.The hemimethylated origins cannot initiate again until the Dam methylase has converted them into fully methylated origins.

Figure 14.35

~ 13 min

delay

What is responsible for controlling reuse of origins?

-- Several mechanisms may be involved: Physical sequestration of the origin

Regulation of methylation by SeqA

Regulation of DnaA binding

by membrane-associated inhibitor

by repression of DnaA transcription by DnaAATP levels

In eukaryotic cells, licensing factor control the re-initiation of replication.

Figure 14.39

The replicator (origin) of S. cerevisiae:

ARS (Autonomously replicating sequence)

圖引用自： Cooper, G. M. (1997) The cell: a molecular approach. ASM Press. Fig. 5.17

Origin recognition complex (ORC)

(Initiation complex)

• ORC is associated with yeast origins throughout the entire cell cycle.

When replication is initiated, Cdc6 and MCM proteins are displaced.

S phase

Cdc6 is rapidly degraded during S phase, preventing re-initiation.

Figure 14.40

DNA Replication





DNA Polymerases



Chromosome in different organisms:

Species Cm. No.Copy No.

FormGenome size (Mb)

Mycoplasma genitalium 1 1 Circular 0.58

E. coli 1 1 Circular 4.6

Agrobacterium tumefaciens 4 1

3 Circular1 Linear

5.67

Saccharomyces cerevisiae 16 1 or 2 Linear 12.1

Arabidopsis thaliana

5 2 Linear 125

Drosophila melanogaster 4 2 Linear 180

Homo sapiens 22 +X/Y 2 Linear 2900

Pro

kary

ote

sEu

kary

ote

s

Extrachromosomal DNA:

DNA in eukaryotic organelles

Plasmid

Viral DNA

Mitochondrial DNA

Chloroplastic DNA

Replication loops always initiate at a unique point, called an origin.

2. Replication is bidirectional.

1. Both DNA strands are replicated simultaneously.

For most eukaryotic and prokaryotic DNAs:

Some extrachomosomal DNAs are replicated unidirectionally or using different replication modes.

2. Replication begins at an origin.


Replication of mitochondrial DNA

Replication of adenovirus DNA

Rolling-circle replication of bacteriophage ssDNA

Replication and transfer of F plasmid

Mitochondrial DNA:

L strand H strand

H strand origin

L strand origin

L H H

RNA primer

HH

Initiation at origin on H

Synthesis of DNA(H as template)

Newly synthesized strand displaces L strand

D loop

Based on Figure 13.11

Replication of the L-strand is initiated when its origin is exposed.

L

H

L

H

L

H

DNA synthesis initiates at left 5’ end.

Single strand is displaced when fork reaches end.

Figure 13.13

duplex origin

Replication of adenovirus DNA:

Terminal proteins enable initiation at the ends of adenovirus DNA.

Figure 13.14

Fig

ure

13.1

5

Rolling-circle replication of bacteriophage circular ssDNA:

(+) strand

5’ 3’

RNA Pol

DNA Pol

LigaseRNA

primer 5’

3’ (-)

(+)

Replicative form

(-)(+) Nick at origin of (+) strand

(-)(+)

(-)(+) (+)

3’-OH

5’-P

The newly synthesized strand displaces old (+) strand.

After 1 revolution displaced strand reaches unit length.Based on Figure 13.16

Continued elongation generates displaced strand of multiple unit lengths

(-)(+)(+)

(-)(+)

(+)

(+)

( replication)

Replication of phage X174:

Replicative form

+

(+) strand

A

Rolling circle replication

-+

A protein nicks the origin and binds to 5’ end.

DNA replication displaces (+) strand.

Replication fork passes origin, A protein nicks DNA & binds to the new 5’ end.

Released (+) strand forms covalent circle.

Based on Figure 13.19

F plasmid (F factor):

~ 100 kb

F cm

F cm

An F plasmid can exist as a free circular plasmid or can integrate into the bacterial chromosome.

The F factor codes for specific pilli that form on the surface of the bacterium.

An F-pilus enables an F+ bacterium to contact an F-bacterium and to initiate conjugation.

Figure 13.21

Donor (F+) Recipient (F-)

TraY/I nick DNA at oriT.

TraY/I multimer migrates around circle, unwinding DNA.

Single strand enters

recipient.

TraY/I

Figure 13.22

Donor Recipient

Figure 13.22

Complementary strands are synthesized.

Donor gap is closed.

Recipient circularizes.

F factor is nicked at oriT.

5’ end leads ssDNA into recipient.

ssDNAs are converted to dsDNA in both bacteria.

Figure 13.23

DNA Replication





DNA Polymerases



Fidelity of replication:

Base selection

Proofreading

Repair systems

(~ 1 error / 1000 cells / generation)

1 mispairing /108 – 1010 bp

Balanced levels of dNTPs

Error rate in E. coli:

DNA Replication

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