chapter 28: dna replication, repair, and recombination copyright © 2007 by w. h. freeman and...

Chapter 28:DNA Replication, Repair, and

Recombination

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Basics of DNA Structure

• The two strands of double stranded DNA run in opposite directions (anti-parallel).

• The purine and pyrimidine bases appended to the deoxyribose-phosphate polymer strands are on the inside of the double helix.

• In the Watson-Crick model Adenine (A) base pairs with Thymine (T) through two H-bonds and Guanine (G) always base pairs with Cytosine (C) through three H-bonds.

Semiconservative Replication

of DNA

Based on studies by Meselson and Stahl. Replication always occurs moving 5'-3'.

A and B DNA

Both are right-handed helices

B = common, 10.4 bp/turn base tilt = 5-6o

A = low humidity, 11 bp/turn base tilt = 11-20o

Ribose ConformationsIn form A the 3' C is up and in form B the 2' C is up. Bases are typically anti to the ribose ring so that base pairing is facilitated.

Ribose AttachmentsRibose in relationship to the groves in ds DNA.

Propeller TwistStudy of a short segment of dsDNA shows non-coplanarity between some base pairs.

DNA, Z FormA left handed helix with alternating GCGC and alternating syn and anti purines.

Three Forms of DNA

DNA Supercoiling

Relaxed DNA in the B form has 10.4 base pairs per turn of the helix. The linear structure below shows the number of turns about the helix axis.

Supercoiling Parameters

The Linking number (L) is the number of times one strand of circular dsDNA passes over the other. L is constant unless the strands break and reform.

The Twist (T) is the number of turns that DNA makes about the duplex axis.

The Writhe (W) is the number of supercoils (turns about the superhelix axis). A clockwise turn = a (+) supercoil and ccw = a (-) supercoil.

So: L = T + W

Circular DNA

Closing the linear DNA seen previously gives relaxed, circular ds DNA with no supercoils so:

L = T + W

25 = 25 + 0

Changing L

Open the circular DNA and unravel two turns. This decreases L by two.

Circular DNA

Closing the DNA makes gives relaxed, circular ds DNA with no supercoils, now:

L = T + W

23 = 23 + 0

Introducing Supercoils

Making two right-handed coils in the previous helix without breaking the strands is equal to a W of –2 and T changes:

23 = 25 - 2

Supercoils

L = T + W

Types of Supercoils

Both types are observed.

Electron MicrographE.Coli has about 5 supercoils per 1000 bp.

DNA Helicase

A helicase unwinds DNA as part of the primosome.

DNA Helicase

Helicase unwinding of dsDNA requires ATP.

Topoisomerase I

Tyr of Topoisomerase I

Tyr of Topo I cleaves one strand of DNA to permit unwinding and a change the linking number (L).

Topoisomerase II

Topo II breaks both strands of DNA and requires ATP.

Topo I breaks only one strand.

DNA Polymerase I

DNA Pol I was the first polymerase isolated. This was obtained from E.coli by Arthur Kornberg and is known as the Kornberg enzyme.

This enzyme has a 5'-3' exonuclease activity that cleaves RNA primers, a 5'-3' polymerase activity that makes DNA and a 3'-5' exonuclease activity that repairs DNA.

The Klenow fragment is the large portion after cleaving off the 5'-3' exonuclease and has been used as a polymerase in lab work.

DNA Polymerase

This is the Klenow fragment of the E.coli enzyme.

DNA Polymerase

Activity

Note the two Mg++ ions binding substrate in this mechanism.

Molecular Shape vs H-Bond

Both of these direct thymine into a DNA strand even though the one cannot form H-bonds.

Conformational Change

When the correct dNTP binds a change occurs resulting in a tight fit for the proper dNTP shape.

E.coli Replication

Three Steps:

1. Initiation: Ori site in E.coli = Ori C This is a 245 bp highly conserved seq.

2. Polymerization: chain elongation in the 5’-3’ direction.

3. Termination: “ter region” is ~350 kbp sequence that is 180o from Ori C.

E.coli OriC Site

The three AT rich sequences on 5' end of OriC are weak, only two H-bonds per bp. To the right of this are five-9 bp sequences that bind DnaA (initiation factor). These five sites have opposing sequences.

Priming Events

DnaA binds to the 9 bp sequences along with ATP and causes opening in the AT rich region. HU a histone-like protein prevents DnaA from binding at sites other than OriC.

When the loop is open DnaB, a helicase, binds at each fork as a complex (DnaB6•DnaC6•ATP6). DnaT assists and this is the pre-priming complex.

DnaB continues to unwind increasing the bubble size displacing DnaA as it moves. SSB binds to ssDNA to prevent annealing. Topo II binds ahead of the fork to relieve stress cause by opening.

Possible DnaA Binding

Proposals suggest that up to 20 or more DnaA are bound and that a nucleosome type structure is formed.

Priming Events

PriA, PriB and PriC enter the bubble along with DnaG (DNA primase). This completes the primosome which makes RNA primers. Topo II and SSB are not part of the primosome. DNA primase does not need a primer to begin synthesis of RNA primers.

The primers (10-30 bp) begin at the center base of any GTT sequence and start a primer about every 1000 bp. Only one primer is needed on the leading strand. After this is made, the primosome moves to the lagging strand.

Replication Events

Two Pol III holoenzymes (DnaE) enter with a few other proteins to complete the replisome. Pol III is an asymmetric dimer that synthesizes DNA from both template strands simultaneously.

Pol III needs a primer to begin synthesis. The leading strand is synthesized continuously and the lagging strand discontinously. Both are synthesized in the 5'-3' direction.

Bidirectional synthesis occurs at both replication forks.

DNA Pol III, the Replicase

Pol III forms a sliding clamp around ds DNA.

This enzyme is the workhorse of replication and is very processive.

Priming DNA Synthesis

A primer is require to start DNA synthesis.

Leading and Lagging Strand Synthesis

Okazaki fragments are made on the lagging strand.

Layout of Participants

General arrangement of replication participants.

Synthesis on both Strands

POL III forms a sliding clamp around ds DNA. This enzyme is very processive.

Lagging Strand Synthesis

The lagging strand loops to enable both Pol III core units to move in the direction of the replication fork.

The lagging strand begins replication at a primer and proceeds until it runs into another Okazaki fragment. At this point the core unit dissociates, the chain shifts to position another primer, the core rebinds and makes another Okazaki piece.

The lagging strand will always be a little behind the leading strand.

Joining Okazaki FragmentsJoining these fragments requires DNA ligase after nick translation has occured.

Steps for Joining Okazaki Fragments

DNA ligase seals the nicks using NAD+ for energy after Pol I has removed RNA. Some organisms use ATP to adenylate the ligase.

E-lys + NAD+ --> AMP-NHlys-E + NMP

5'-p-DNA + AMP-NHlys-E --> AMP-5'P-DNA + E-lys

AMP-5'P-DNA + 3'OH-DNA --> DNA(sealed) + AMP

E.coli Termination

The E.coli ''ter region'' is a ~350 kbp sequence 180o from Ori C. It contains seven sequences, TerA to TerG, which are binding sites for ''tus'', terminator utilization substance. The Ter sequences are ~ 20 bp long and contain the conserved sequence 5'-GTGTGTTGT-3'. When tus is bound replication stops by blocking the helicase.

G F B C A D E

5' ------------------------------------™------™------™------- 3'

Ter region (~4.5 min on clock face)

E.coli Termination

The clockwise replication is stopped at ter B,C,F or G and counterclockwise replication at ter A,D or E. The process is complete when synthesis from the opposite direction reaches the stopped strand.

At this point, the two new DNAs are intertwined and Topo II mediates unraveling these by cleavage and reassembly.

Replication Comparison

Procaryotic Eucaryotic

speed 1000 b/sec 50 b/sec

Okazaki 1000 b 100-200 b

primer ~30 b ~3-5 b

ori sites single multiple

Pol nuclease no nuclease

TelomeresA sequence at the ends of linear eucaryotic chromosomes that helps stabilize the chromosome. In humans this is a repeat of AGGGTT and is added to the ends of the chromosome by the enzyme telomerase. Telomerase, a reverse transcriptase, contains an RNA component that codes for the telomere.

TelomereSynthesis

Telomerase has an RNA template.

TelomereSynthesis

Mutations

Fidelity is good ~ 1:109. Both Pol I & III have proofreading/correction capability.

Mutation: permanent alteration, damage that escapes repair

Substitutions (silent): Transversion replaces pur with pyr or pyr with pur. Transition replaces pur with pur or pyr with pyr.

Frameshift (lethal): Addition adds an extra base, elongates DNA. Deletion removes a base and shortens DNA. These change every triplet.

DNA Damage

Examples of sources of damage.

Deamination: nitrous acid.

Methylation: N-Me-N-nitrosourea or Dimethylnitrosamine (Me2N-N=O)

Intercalation: Polynuclear aromatic hydrocarbons

Uv damage: Photodimerization of T, loss of base (AP formation), phosphodiester cleavage.

Strand breakage: uv or x-rays

OxidationMay lead to errors in H-bonding association and base pair mismatch.

DeaminationNaNO2 + HCl generates nitrous acid, which converts a primary aromatic amine to a carbonyl.

Uv Dimerization

This is a photolytic 2 + 2 cycloaddition reaction.

Correction by Pol III

Pol III has exonuclease activity that allows correction of base pairing errors. Pol I also has proofreading capability. DNA is the only biopolymer repaired.

Direct Repair

Direct Repair:

Photolyase, a photoreactivating enzyme that reverses a uv induced thymine dimer (needs vis).

Insertase, is an enzyme that can replace a specific base at AP site.

O6-methylguanine methyltransferase is a suicide enzyme (TON = 1) that transfers methyl from O6-methylguanine to a Cys on the enzyme and as a result loses activity.

Excision Repair

Excision Repair: (1. base and 2. nucleotide)

Base: Deamination or methylation may modify a base. A glycosylase (AlkA) recognizes and cleaves the modified base to produce an AP site. AP endonuclease cleaves the strand, Pol I fills and DNA ligase seals the gap. Note: thermal effects spontaneously produce AP sites ~ 5 x 103 per day.

Nucleotide or general: Exinuclease (excision repair endonuclease) cleaves both sides of the damaged site making a ssDNA gap, Pol I fills and DNA ligase seals the gap.

Glycosylase

AlkA in E.coli.

About 20 of these enzymes are known.

There is one specific for uracil.

Base ExcisionRepair

Uracil Glycosylase:

Deamination of cytosine gives uracil. This error is corrected by base excision repair. Leaving uracil in place would produce a C-G to U-T transition.

Nucleotide ExcisionRepair

Mismatch Repair

Mismatch Repair:

Focus is primarily on correcting non-Watson-Crick base pairs called Hoogsteen base pairs. C-C is least responsive to repair and T-G is the easiest. The newly made strand is corrected to match the template. This involves three proteins: MutS, Mut L and Mut H. MutS binds to the mismatch, then Mut H binds to 6-MeA in GATC of the parent near the mismatch and cleaves. MutL links MutS and MutH. Exonuclease I removes the mismatch segment 3’-5’. Pol III comes in and fills in from the template and DNA ligase seals the gap.

Mismatch Repair

SOS Response

SOS Response:

Repair in this instance almost always results in inaccurate or “error prone” production of a daughter strand due to polymerization through an unreadable template. As a result, a mutant is produced. The error(s) produced in the daughter strand may or may not be lethal, however this is the only path available. The alternative is cell death caused by incomplete replication.

SOS Response

SOS Response:

A protein, LexA, normally binds to the SOS box to prevent expression. Major DNA damage results in RecA protein binding to the ssDNA and this in turn promotes autolysis of LexA. The SOS box is now unprotected and is expressed. The resulting SOS proteins are capable of repairing large segments of DNA, even filling in gaps where bases are missing.

RecombinationThis is sometimes called post-replication repair.

End of Chapter 28

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition