Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter TenBiosynthesis of Nucleic Acids: Replication

Replication of DNA

• Naturally occurring DNA exists in single-stranded and double-stranded forms, both of which can exist in ________ and ________ forms

• Difficult to generalize about all cases of DNA replication

• We will study the replication of ________ ________ ________ DNA and then of ________ ________ ________ DNA• most of the details we discuss were first investigated

in ________, particularly ________ ________

Flow of Genetic Information in the Cell

• Mechanisms by which information is transferred in the cell is based on the “________ ________”

Prokaryotic Replication

• Challenges in duplication of ________________ double-stranded DNA• achievement of continuous ________ and

separation of the two DNA strands• ________ of ________ portions from attack by

________ that attack single-stranded DNA • synthesis of the DNA ________ from one 5’ -> 3’

strand and one 3’ -> 5’ strand• efficient protection from ________ in replication

Prokaryotic Replication (Cont’d)

• Replication involves ________ of the two original strands and ________ of two new daughter strands using the ________ strands as ________

• _________________ replication:_________________ replication: each daughter strand contains one template strand and one newly synthesized strand

• Incorporation of isotopic label as sole _________ source (15NH4Cl)

• Observed that 15N-DNA has a higher density than 14N-DNA, and the two can be separated by density-gradient ______________

Which Direction does Replication go?

• DNA double helix unwinds at a specific point called an ____________________________________________________________

• Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is _______________ _______________ in most organisms

• At each origin of replication, there are two _________ _________ __________________________, points at which new polynucleotide chains are formed

• There is ______ origin of replication and ________ replication forks in the circular DNA of prokaryotes

• In replication of a _______________ _______________ chromosome, there are several origins of replication and two replication forks at each origin

DNA Polymerase

• DNA is synthesized from its 5’ -> 3’ end (from the 3’ -> 5’ direction of the template)

• the ________________ strand strand is synthesized continuously in the 5’ -> 3’ direction toward the replication fork

• the ________ ________ strandstrand is synthesized __________ __________ (________ fragments)________ fragments) also in the 5’ -> 3’ direction, but away from the replication fork

• lagging strand fragments are joined by the enzyme DNA DNA ______________________

DNA Polymerase

DNA Polymerase Reaction

• The 3’-OH group at the end of the growing DNA chain acts as a ______________________________.

• The ______________ ______________ adjacent to the sugar is attacked, and then added to the growing chain.

Properties of DNA Polymerases

• There are at least _______ types of DNA polymeraseDNA polymerase (Pol) in E coli, _______ of which have been studied extensively

Function of DNA Polymerase

• DNA polymerase function has these requirements:• all four deoxyribonucleoside triphosphates: dTTP,

dATP, dGTP, and dCTP• Mg2+

• an _________ - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication

• DNA-Pol I: repair and patching of DNA• DNA-Pol III: responsible for the polymerization of the

newly formed DNA strand• DNA-Pol II, IV, and V: proofreading & repair enzymes

Supercoiling and Replication

• DNA DNA _________ (class II topoisomerase) catalyzes rxn involving relaxed circular DNA:• creates a _______ in

relaxed circular DNA• a slight unwinding at

the point of the nick introduces _______

• the nick is _______• The energy required for

this process is supplied by ____________________

Replication with Supercoiled DNA

• Replication of supercoiled circular DNA• DNA gyraseDNA gyrase has different role here. It has different role here. It introduces a

nick in supercoiled DNA• a _______ point is created at the site of the _______• the gyrase opens and _______ the swivel point in

advance of the replication fork• the newly synthesized DNA automatically assumes

the ___________ form because it does not have the nick at the swivel point

• _______, a helix-destabilizing protein, promotes unwinding by binding at the replication fork

• single-stranded binding (SSB) protein _______ single-stranded regions by _______ tightly to them

Primase Reaction

• The primase reaction• RNA serves as a _______ in DNA replication• _______ activity first observed in-vivo.• Primase -Primase - catalyzes the copying of a short stretch of

the DNA template strand to produce RNA primer sequence

• __________ and _________ of new DNA strands• begun by DNA polymerase III• the newly formed DNA is linked to the 3’-OH of the

RNA primer• as the replication fork moves away, the RNA primer is

removed by DNA polymerase I

Replication Fork General Features

DNA Replication in Prokaryotes

• DNA synthesis is bidirectional• DNA synthesis is in the 5’ -> 3’ direction

• the leading strand is formed continuously• the lagging strand is formed as a series of Okazaki

fragments which are later joined• Five DNA polymerases have been found to exist in E. coli

• Pol I is involved in synthesis and repair • Pol II, IV, and V are for repair under unique conditions• Pol III is primarily responsible for new synthesis

DNA Replication in Prokaryotes

• Unwinding• DNA gyrase introduces a swivel point in advance of the

replication fork• a helicase binds at the replication fork and promotes

unwinding• single-stranded binding (SSB) protein protects exposed

regions of single-stranded DNA• Primase catalyzes the synthesis of RNA primer• Synthesis

• catalyzed by Pol III• primer removed by Pol I• DNA ligase seals remaining nicks

Proofreading and Repair

• DNA replication takes place only once each generation in each cell

• Errors in replication (mutations) occur spontaneously only once in every 109 to 1010 base pairs

• Can be lethal to organisms

• Proofreading - the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication (Figure 10.10)

• Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 104 to 105 base pairs

Proofreading Improves Replication Fidelity

• ____________________: catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides

• ______________________: Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity

• ______________________: enzymes recognize that two bases are incorrectly paired, the area of mismatch is removed, and the area replicated again

• ______________________: a damaged base is removed by DNA glycosylase leaving an AP site; the sugar and phosphate are removed along with several more bases, and then Pol I fills the gap

DNA Polymerase Repair

Mismatch Repair in Prokaryotes

• Mechanisms of mismatch repair encompass:

Eukaryotic Replication

• Not as understood as prokaryotic. Due in part to higher level of complexity.

• Cell growth and division divided into phases: M, G1, S, and G2

Eukaryotic Replication

• Best understood model for control of eukaryotic replication is from yeast.

• DNA replication initiated by chromosomes that have reached the G1 phase

Eukaryotic DNA Polymerase

• At least 15 different polymerases are present in eukaryotes (5 have been studied more extensively)

Structure of the PCNA Homotrimer

• PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp ().

The Eukaryotic Replication Fork

The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Differences summarized in Table 10.5.

Telomerase and Cancer (Biochemical Connections)

• Replication of linear DNA molecules poses particular problems at __________________ of the molecules

• Ends of eukaryotic chromosomes called ______________________:

__________________________________________

• See figures on p. 282-283