FCH 532 Lecture 12

Chapter 30: DNA replication

Exams graded: average was 69

Table 11.1 Comparison of E. coli DNA polymerases

• Primary replicating enzyme in E. coli cells is thought to be DNA pol III: faster, more complex structure.

• DNA pol I and II probably serve in editing and repair of DNA.

A DNA Replication Paradox:

1. DNA strands must be copied in both directions during replication.

2. However, all known DNA polymerases catalyze chain formation in the 5’3’ direction.

Paradox resolved by Reiji Okazaki and coworkers:

• Observed short 1000-2000 nucleotide fragments during replication.

• Small fragments are covalently joined in later steps to form completed lagging daughter strand.

Semidiscontinous replication

• If DNA strands are extended 5’ to 3’, how can the antiparallel strand by simultaneously replicated past the replication fork?

• Okazaki used pulse-chase experiment.

• E. coli DNA pulse-labeled 30 s with of [3H]thymidine that changes the sedimentation coefficient of newly made DNA from 7S to 11S (Okazaki fragments).

• Okazaki fragments: 1000-2000 nt in prokaryotes; 100-200 nt in eukaryotes

• Follow [3H]thymidine pulse with transfer to unlabeled medium (pulse-chase)-the resulting radioactively labeled DNA sediments at rate that increases with time the cells grow in unlabeled medium.

• Okazaki fragments incorporated into the larger DNA.

Figure 30-5 Semidiscontinuous DNA replication. In DNA replication, both daughter strands (leading strand red, lagging strand blue) are synthesized in their 5 3

directions.

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RNA Primers

• Need a 3’-OH group to extend DNA chain.

• Analysis of Okazaki fragments revealed that they have short (1-60 nt) RNA segments complementary to the template DNA.

• RNA primers catalyzed by 2 enzymes:

• RNA polymerase, large (459 kD), mediates transcription, rifampicin sensitive.

• Primase (DnaG), small (60 kD), rifampicin resistant.

• Rifampicin inhibits only leading strand synthesis, therefore, primase initiates Okazaki fragments.

• Leading strand can be mediated in vitro by both, is better when both enzymes present.

Figure 30-7 Priming of DNA synthesis by short RNA segments.

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Figure 11.8 Closeup of a replication fork showing initiation of the continuous leading strand and the discontinuous, lagging strand (Okazaki fragments)

• All known DNA polymerases catalyze chain formation in the 5’ 3’ direction.

• DNA strands must be copied in both directions!

Figure 11.9a Complete scheme showing sequential steps of replication process.

1. Helicase (unwinding protein / rep protein) recognizes and binds origin of replication.

• Catalyzes separation of the two DNA strands by disrupting H-bonding between base pairs.

• Endothermic reaction is coupled to hydrolysis of ATP.• DNA gyrase (a topoisomerase) assists in unwinding by inducing

supercoiling.

Figure 11.9b Complete scheme showing sequential steps of replication process.

2. Single-stranded DNA binding proteins (SSB) bind exposed strands of DNA.

• Protect it from hydrolytic cleavage of phosphodiester bonds.

Figure 11.9c Complete scheme showing sequential steps of replication process.

3. Primer synthesis: Short complementary stretch of RNA (4-10 bases) is synthesized by primase enzyme.

• Primer with free 3’-OH is required by DNA pol III to start 2nd strand synthesis.

• RNA primer is later degraded by 5’->3’ exonuclease action of DNA pol I, RNaseH enzymes.

Figure 11.9d Complete scheme showing sequential steps of replication process.

4. DNA synthesis by DNA pol III begins, extending leading and lagging strands.

• DNA synthesis continues until it meets next fragment.

Figure 11.9e Complete scheme showing sequential steps of replication process.

5. RNA primers are removed by 5’3’ exonuclease action of DNA polymerase I, small gaps are filled in by DNA polymerase I.

Figure 11.9f Complete scheme showing sequential steps of replication process.

6. Final gap between new strands is closed by DNA ligase enzyme.

• Requires ATP to join 3’ OH on one fragment and 5’ phosphate on second fragment.

Figure 11.10 The DNA ligase-catalyzed reaction to close the final phosphodiester bond in newly synthesized DNA.

ATP is required as a source of energy for this endergonic reaction.

DNA replication proteins

Eukaryotic DNA polymerases

• Several eukaryotic DNA polymerases also known:

• found only in nucleus, requires template and primer.

• also found in nucleus

• found in mitochondria

Table 6–1 Error Rates in DNA synthesis

If an incorrect nucleotide is added to a growing strand, the DNA polymerase will cleave it from the strand and replace it with the correct nucleotide before continuing.

DNA Pol I is a proofreading enzyme

• 3 active sites on the same enzyme: DNA polymerase activity, a 3’ 5’ exonuclease and a 5’ 3’ exonuclease.

• 5’ 3’ exonuclease activity is independent of the 3’ 5’ exonuclease and polymerase activity.

• Pol I can be cleaved by trypsin into two fragments: larger C-terminal fragment is called the Klenow fragment (KF) and contains the polymerase and 3’ 5’ exonuclease activity. The N terminal fragment contains the 5’ 3’ exonuclease activity.

• Pol I is a processive enzyme; it catalyzes a series of successive polymerization events (20 or more) without releasing the template.

Figure 11.7a Exonuclease activity of DNA polymerase I

• (a) 3’5’ exonuclease activity

DNA polymerase I acts as a proofreading and repair enzyme by catalyzing hydrolytic removal of mismatched bases.

Figure 11.7b Exonuclease activity of DNA polymerase I

• (b) 5’3’ exonuclease activity

DNA polymerase I acts as a proofreading and repair enzyme by catalyzing hydrolytic removal of mismatched bases.

Figure 30-10Schematic diagram for the nucleotidyl transferase mechanism of DNA polymerases.

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2 metal ions (Mg2+)

B ligands the phosphates of dNTP

A bridges Pa with the primer’s 3’OH for in-line nucleophilic attack.

Pol I

• 3’-5’ exonuclease functions in editing DNA.

• Crystal structure shows a 12 nt “template” strand (5’-TGCCTCGCGGCC-3’) and a 7 nt “primer” strand (3’-GCGCCGG-5’) that is complementary to the 3’ end of the template strand and a 2’,3’-epoxy-ATP.

• Forms distorted segment.

O

2’,3’-epoxy-ATP

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template

primer

Pol I

• A second primer strand is also present that base pairs with the 5’-terminal nucleotides but has a single T overhang on the template strand and a 3 nucleotide overhang on the primer strand.

• The 3’-terminal nucleotide of the primer strand is bound at the 3’-5’ exonuclease site (editing complex).

• The 3’ end of the primer strand is in competition to go between the active site and editing site. If properly base paired it goes to the active site, otherwise it goes in the editing site.

Figure 30-11Probable sequence of the double-stranded DNA seen in the X-ray structure of KF.

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Other Pol I activities

• Pol I functions to repair DNA by simultaneously filling in single-strand gaps formed during endonucleolytic cleavage of DNA.

• The 5’-3’ exonuclease and polyerase activities can replace nt on the 5’ side of a single strand nick.

• These reactions move the nick toward the 3’ end of the DNA strand (nick translation).

• Can be used to create radiolabeled DNA.

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Figure 30-12 Nick translation as catalyzed by Pol I.

Other Pol I activities

• The 5’-3’ exonuclease activity also removes RNA primers at the 5’ ends of newly synthesized DNA.

• DNA polymerase fills in gaps.

• Used temperature-sensitive E. coli mutants to demonstrate the function.

• Essential for survival.

DNA Pol III is the replicase

• Pol III core has multiple subunit composition. The polC gene encodes the polymerase function.

• Has a 3’ 5’ exonuclease editing function.• 5’ 3’ exonuclease activity can only act

on single stranded DNA.

• Pol III core has processivity for only 10-15 residues.

• Needs the -subunit in the presence of the complex for maximum processivity (>5000 residues).

• -subunit forms a sliding clamp on the DNA.

Table 30-2 Components of E. coli DNA Polymerase III Holoenzyme.

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Figure 30-13a X-Ray structure of the subunit of E. coli Pol III holoenzyme. Ribbon drawing.

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Figure 30-13b The subunit of E. coli Pol III holoenzyme. Space-filling model of sliding clamp in

hypothetical complex with B-DNA.

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DNA is unwound by Helicases

• Helicases are involved in DNA replication, recombination, repair and transcription termination, RNA splicing, and RNA editing.

• Translocate on one strand of double-helical nucleic acid to unwind the double helix using the energy of hydrolysis of NTPs.

• Classified by their direction: 5’-3’ and 3’-5’ helicases.

Table 30-3 Unwinding and Binding Proteins of E. coli DNA Replication.

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Helicases

• DnaB-hexameric helicase, 5’-3’ translocation along the lagging strand, hydrolyzes ATP. Cannot use UTP. No crystal structure.

• T7 gene 4 helicase/primase-2-tiered hexagonal ring with N-terminal domains containing the primase activity and C-termini carry out the helicase activity. Uses dTTP (also dATP and ADP). Crystal structure known (Hexameric C-termini similar to DnaB).

Figure 30-14 Unwinding of DNA by the combined action of DnaB and SSB proteins.

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Figure 30-15Electron microscopy–based image reconstruction of T7 gene 4 helicase/primase.

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Figure 30-16X-Ray structure of the helicase domain of T7 gene 4 helicase/primase.

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Helicases

• Rep helicase- monomeric enzyme, but active form is a dimer with 3’-5’ translocation.

• Shows negative cooperativity, one subunit binds to ssDNA and inhibits the binding of the other subunit to ssDNA(binds to dsDNA).

• Enzyme operates through “active/rolling” mechanism.

• Unwinds DNA by walking up strand in ATP dependent manner.

• PriA -is a similar monomeric enzyme with 3’-5’ translocation

Figure 30-17Active, rolling mechanism for DNA unwinding by Rep helicase.

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Figure 30-18X-Ray structure of Rep helicase in complex with dT(pT)15 and ADP.

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ADP

ssDNA

Closed Open •Two domains (1 and 2)

•Each domain is in 2 subdomains (1A, 2A-N-terminal subdomain)

•Change occurs in domain 2B from open to closed

Figure 30-19X-Ray structure of the N-terminal 135 residues of E. coli SSB in complex with dC(pC)34.

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•SSB prevent ssDNA from reannealing to form dsDNA

•Homotetramer

•2 major forms (subscript is # of nt)

•(SSB)35 in which 2 of of the four subunits interact with ssDNA

•(SSB)35 shows unlimited cooperativity

•(SSB)65 in which all 4 of the subunits interact with ssDNA; limited cooperativity.

ssDNA

Figure 30-20The reactions catalyzed by E. coli DNA ligase.

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• Can use either NAD+ converted to NMN+ +AMP or ATP to PPi and AMP.

1. The adenylyl group of NAD+ is transferred to -amino group of Lys to form phosphoamide addict (unusual).

2. Adenylyl group of activated enzyme is transferred to 5’-phosphoryl terminus of nick to form adenylylated DNA (AMP linked to 5’nucleotide via PPi.

3. DNA ligase catalyzes formation of phosphodiester bond by attack of the 3’-OH group on the 5’-nucleotide.

Primase

• Closely associated with DNA helicase (T7 gene 4 helicase/primasehas both domains)

• E. coli primase (DnaG) forms noncovalent complex with DnaB

• Primase reverses its direction in order to synthesize RNA primer in 5’-3’ direction.

• 3 domains: N-terminal Zn2+ binding domain, central catalytic domain with Mg2+ , C-terminal domain interacts with DnaB.

Figure 30-22X-Ray structure of E. coli primase.

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