Lecture 4: DNA repair & recombination

Proof-reading

Repair of base mismatch occurring during replication

Types of repair of neo-synthesised DNA

mismatch repair, base-excision repair, nucleotide-excision repair, direct repair

What happens when base mismatch occurs during replication?

• Different geometry

• Directly and immediately removed by DNA polymerase during replication

• 3’ → 5’ exonuclease activity of DNA pol I (or ε, δ)

How is parent strand identified?

Parental DNA is methylated in first few minutes after replication

Methylation in bacteria

• Dam methylase (bacterial DNA methylase) recognises the GATC sequence

• Adds the -CH₃ group at the N⁶ positions of the Adenine (S-adenosyl methionine is the alkylating agent)

Methylation in eukaryotes

In eukaryotes, methylation occurs in the Cytosine that precedes a Guanine, in a 5’CpG

Early phases in the mismatch repair in E. coli

1. MutS scans DNA and forms a clamp-like complex when it encounters a lesion (complex binds to all mismatched bases, except C-C).

2. MutL forms a complex with MutS.

3. The MutSL complex slides along the DNA to find a hemimethylated GATC sequence.

4. MutH binds to MutL, forming MutSLH complex which moves in either direction at random.

5. When a hemimethylated GATC sequence is found, MutH (an endonuclease) is activated.

6. Activated MutH cleaves the unmethylated strand on the 5' side of the G in the GATC, marking the strand for repair.

Later phases of mismatch repair

Mismatch repair system in eukaryotes

homolog of MutS → MSH2, MSH3, MSH6

homolog of MutL → heterodimer MLH1 + PMS1

MutH → no homolog in eukaryotes

MSH2-6 heterodimers bind to….?

Single bp mismatches and less to slightly longer mismatches

MSH2-MSH3 dimer binds…?

Longer mismatches (2-6 bp) in many organisms

Base-excision DNA repair

Excision of bases recognised as DNA components

• e.g. uracil which could replace cytosine by deamination and pair to adenine or hypoxanthine which could replace adenine by deamination and pair to cytosine, as a guanine

Why does DNA contain Thymine and not Uracil like RNA

• Uracil spontaneously forms (slow reaction) by deamination of cytosine, therefore it would become not recognisable from other U bases, if they were “physiologically” allowed.

• Thus, when U forms in the DNA it is recognised and eliminated

Enzymes involved in base-excision DNA repair of an AP site

• DNA glycosylase

• AP endonuclease + helicase

• DNA polymerase I

• DNA ligase

DNA glycosylase

Cleaves bond between base and ribose

AP endonuclease

Cuts phosphodiester bond at AP site

How many types of uracil DNA glycosylases in bacteria and humans?

• Bacteria: Only 1 type

• Humans: At least 4 types

UNG

Eliminates occasional U instead of T in DNA at replisome

hSMUG1

Removes any U in ssDNA during replication or transcription

TDG

Removes U paired with G by deamination by C (deaminated C is U)

MBD4

Removes T paired with G by 5-methylcytosine (deaminated 5-methyl is T)

Nucleotide-excision repair

• For DNA lesions that large distortions in the helical structure of DNA

• Critical pathway for the survival of all free-living organisms

Excinuclease

• Multi sub-unit enzyme

• Hydrolyses 2 phosphodiester bonds, one on either side of the distortion caused by the lesion

Excinuclease in E. coli features

• 5th bond 3’ side, 8th bond on 5’ side (+1-2 damaged nucleotides)

• 12-13 nucleotide fragments

Excinuclease in eukaryotes

• 6th on 3’ side, 22nd bond on 5’ side (+ 1-2 damaged nucleotides)

• 27-29 nucleotide fragments

Nucleotide-excision repair in E. coli

E. coli excinuclease → DNA helicase (13 mer) → DNA pol I → DNA ligase

Nucleotide-excision repair in humans

Human excinuclease → DNA helicase (29 mer) → DNA pol ε → DNA ligase

ABC excinuclease system subunits

UvrA, UvrB, UvrC

Mechanism of the ABC excinuclease system

• UvrA dimer (ATPase) scans DNA and binds to a lesion

• UvrB binds to UvrA

• If a lesion is found UvrA dissociates and leaves a tight UvrB-DNA complex

• UvrC binds to UvrB

• UvrB makes an incision at the 5th bond on the 3’ side of the lesion

• UvrC makes an incision at the 8th bond on the 5’ side of the lesion

• 12-13mer is removed by UvrD helicase

• Gap repaired by DNA Pol I (E. coli)

Nucleotide-excision repair is the primary repair route for…?

cyclobutane pyrimidine dimers, 6-4 photoproducts, benzopyrene-guanine

How is nucleotide-excision repair different in eukaryotes than bacteria?

Similar mechanism but at least 16 proteins with no similarity to ABC excinuclease system are involved

Direct repair

Lesion is corrected in place

How is O⁶-methylguanine repaired? What type of reaction is it?

• Direct repair by O⁶-methylguanine methyltransferase

• -CH₃ removed → Guanine formed

• Non-enzymatic reaction (protein methylation permanently inactivates the methyltransferase)

In which situations is DNA repair not possible by proofreading, mismatch repair, base-excision repair, nucleotide-excision repair or direct repair?

• Unrepaired lesion halts replication fork and generates a ssDNA

• Strand break halts the replication fork (one of the arms is lost) and the fork collapses

What can be used to correct:

• Unrepaired lesion halts replication fork and generates a ssDNA

• Strand break halts the replication fork and the fork collapses

• DNA recombination

• Error-prone TLS (trans-lesion DNA synthesis)

TLS is part of _________ in bacteria

SOS response

What proteins are involved in TLS?

UvrA, UvrB, UmuC, UmuD

TLS in bacteria

• UmuD cleaved in SOS-regulated process to UmuD’

• UmuD’ binds to UmuC and RecA to form DNA pol V

DNA pol V is made of…?

UmuD’₂-UmuC-RecA

DNA pol V function

• Can replicate past many of the DNA lesions that would normally block replication

• Proper base pairing is impossible → error-prone system (desperate strategy)

Like DNA pol V, what other enzyme is also very error-prone?

DNA pol IV

What is the error rate of DNA pol IV and V

Reduces fidelity to 1 error in 1000 nt

Which enzymes involved in TLS in mammals?

Low-fidelity polymerases:

• DNA polymerase η (all eukaryotes)

• DNA polymerases β, ι, λ

DNA polymerase η

• Promotes TLS across cyclobutane T-T dimers

• Few mutations occur since the enzyme preferentially inserts A-A

DNA polymerases β, ι, λ

• After the action of glycosylase and AP endonuclease, they remove the abasic site and randomly fill the very short gap.

• The very short length of DNA synthesised minimises the mutation rate

Define DNA recombination

The rearrangement of genetic information within and among DNA molecules

Types of DNA recombination

• Homologous genetic recombination

• Site-specific recombination

• DNA transposition

Homologous genetic recombination

• Involves genetic exchanges between any 2 DNA molecules or segments that share an extended region of nearly identical sequence

• Includes a process to repair ds breaks in DNA (NHEJ)

Site-specific recombination

Exchanges occur only at specific DNA sequences

DNA transposition

Involves a short segment of DNA capable of moving from one location to another (jumping genes)

Homologous recombination is primarily a DNA repair process in …?

bacteria (recombination DNA repair)

Homologous recombination purposes

• Reconstruction of replication forks (stalled or collapsed)

• Increasing genetic diversity during conjugation

Recombinational DNA repair at a collapsed replication fork

1. The 5’ ending strand at the break is degraded

2. ss 3’-extension is created

3. 3’-extension invades and pairs with its complementary strand in the adjacent duplex

4. Migration of the branch creates a Holliday intermediate

5. Specialised nucleases resolve the Holliday intermediate

6. Ligation restores a viable replication fork and replication resumes

Holliday intermediate

A branched nucleic acid structure that contains 4 double-stranded arms joined together in several possible conformations

What is responsible for homologous recombination in E. coli?

RecBCD nuclease/helicase system

RecD

5’ → 3’ helicase

RecB

3’ → 5’ helicase, nuclease (degrades both DNA strands as they unwind)

Describe homologous recombination in E. coli

• Helicases (RecB and RecD) halt at CHI sequences

• CHI sequence binds tightly to RecC

  • Creation of ss 3’-extension

    • Degradation of 3’ ending strand is greatly reduced

    • Unwinding and degradation of 5’ ending strand is increased

• RecA recombinase is loaded

  • Promotes strand invasion + strand exchange reactions

• RuvAB complex promotes branch migration

  • Formation of Holliday intermediate

• RuvC specialised nucleases cleave and resolve the Holliday intermediate

• Nicks are sealed by DNA ligase

CHI sequence

5’-GCTGGTGG-3’

RuvA

Binds to the Holliday intermediate

RuvB

• Hexameric, translocase

• 2 RuvB hexamers bind to opposite arms of the Holliday intermediate

  • Propel DNA outward in a reaction coupled to ATP hydrolysis

  • The branch thus moves

RuvC

• Binds to RuvAB and cleaves the Holliday intermediate on opposite side of the junction

  • The 2 contiguous DNA arms remain in each product

What happens after the replication fork reassembles?

Origin-independent restart of replication

Replication restart primosome

PriA, PriB, PriC, DnaT, DnaC, DnaB, DnaG

Describe origin-independent restart of replication

• 4 proteins (PriA, PriB, PriC, DnaT) act with DnaC to load DnaB helicase onto the reconstructed replication fork

• DnaG primase synthesises an RNA primer

• DNA pol III reassembles on DnaB to restart DNA synthesis

Functions of homologous recombination in eukaryotes

• Repair of several types of DNA damage

• Transient physical link between chromatids promoting orderly segregation of chromosomes at the first meiotic division

  • Enhancement of genetic diversity in a population

Where does homologous recombination take place in eukaryotes?

Chiasmata

How are chiasmata formed?

In prophase I, just before the first meiotic division, the 2 sets of chromatids (each have replicated dsDNA molecule) align to form tetrads, held together by covalent links at homologous junctions (chiasmata).

How is proper segregation ensured in dividing cells?

Transient associations between homologs creates a tension that allows proper segregation when spindle fibers pull them towards the poles of dividing cells in the first meiotic division

What is NHEJ and when is it needed?

Non-homologous end joining

• Ds breaks occurring when recombinational DNA repair is not feasible

• e.g. when the break occurs in phases in which DNA is not replicating + no sister chromatids

Disadvantages of NHEJ

• Highly mutagenic; smaller genomes cannot afford it → they privilege homologous recombination

• Does not preserve the original DNA sequence

Ku70-Ku80

Heterodimer binding the DNA ends (scaffold to assemble other molecules)

DNA-PKcs

Protein kinase

Artemis

Nuclease

P-Artemis

Endonuclease

Describe NHEJ

• DNA ends bound by Ku70-Ku80

• Ku70-Ku80 recruits DNA-PKcs and Artemis

• The 2 DNA ends are synapsed (held together)

• DNA-PKcs autophosphorylates and phosphorylates Artemis

• P-Artemis is activated, and removes 5’- or 3’-ss extensions or hairpins at the ends

• A helicase separates the 2 ends and strands from different ends are annealed at regions of short complementarity.

• Artemis removes any unpaired DNA.

• Small gaps are filled by either DNA Polymerase μ or λ

• The nicks are sealed by a protein complex composed of XRCC4, XLF and DNA ligase IV

NHEJ consequence

Broken ends are kept close together by chromatin structures to avoid joining far apart areas; could lead to deleterious rearrangements

Where does site-specific recombination occur?

In every cell

Role of site-specific recombination

• Gene expression regulation

• Programmed DNA rearrangements in embryo development

• Replication cycle of viral, plasmidic DNA

Site-specific recombination requires…?

• Recombinase (2 classes: Tyr or Ser in active site)

• Short (20-200 bp), unique DNA sequences (recombination site)

• One or more auxiliary proteins (timing of reaction)

Tyr-class recombinases

• 2 separate recombinases on recombination site within same or on different DNA molecules

• One strand in each site is cleaved at a specific point within the site.

• Recombinase covalently links to the DNA (phospho-tyrosine bond).

• Transient protein-DNA linkage preserves the phosphodiester bond lost in DNA cleavage, so ATP not necessary in following steps.

• Cleaved DNA is re-joined to new partners at the expense of P-Tyr bond (formation of Holliday intermediate)

  • Isomerization: Process repeated at a second point within each of the 2 rec sites

Recombinases act as both ______ and ______

site-specific endonuclease and ligase

Ser-class recombinases

Both strands of each recombination site are cut simultaneously and rejoined to new partners without Holliday intermediates

In both Tyr- and Ser- classes…?

The exchange is always reciprocal, precise, and regenerates the recombination sites when completed

What happens if 2 recombing sites align in the opposite orientation during the recombinase reaction?

Same DNA molecule: inversion

What happens if 2 recombing sites align in the same orientation during the recombinase reaction?

1 DNA molecule: deletion

2 DNA molecules: insertion

Transposons

Segments of DNA that can move (“jump”) from one place (donor site) to another in the same or a different (target site)

Where are transposons found?

In all cells

Is transposition a random process?

More or less, but it is tightly regulated (it could kill the cell)

Is DNA sequence homology required for transposons?

No

What are the classes of transposons in bacteria?

1. Insertional sequences (simple transposons)

2. Complex transposons

Insertional sequences

Sequence required for transposition + genes for transposases

Complex transposons

Also contain one or more genes (relevance for the transmission of antibiotic resistance among bacteria)

Transposase definition + mechanism

• Enzyme promoting transposition

• Makes staggered cuts in the target site

Transposase binding sites

Short repeated sequences (short terminal repeats, 5-10 bp)

What happens when a transposon is inserted into target site?

Transposition results in the duplication of flanking DNA sequence at target site

Direct transposition

• Cut on each side of transposon and excision (leaves ds break that needs repair at the donor site)

• Staggered cut at the target site → transposon is inserted and DNA replication fills the gaps to duplicate the target-site sequence

Replicative transposition

• Transposon is replicated (a copy is left at the donor site)

• An intermediate cointegrate forms

  • Cointegrate consists of the donor region covalently linked to DNA at the target site and contains 2 copies of the transposon, with the same relative orientation

  • Possible site-specific recombination in cointegrate intermediate

Eukaryotic transposons

• Structurally similar to bacterial transposons

• Similar transposition mechanisms

• In some cases transposition involves an RNA intermediate

Immunoglobin genes assemble by…?

Recombination