Molecular Biology: Chapter 5 — Damage and repair

5.1 What the DNA suffers #

5.1.1 Deamination, depurination, dimers, mismatches, and breaks #

Water and ordinary metabolism damage DNA in an undamaged cell, which is why repair is constitutive.

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Deamination of cytosine produces uracil, which pairs as thymine and would cause a C·G to T·A transition if left. 5-Methylcytosine deaminates to thymine, a normal base, so the product is a T·G mismatch and a harder call. Methylated CpG is therefore a mutation hotspot (Chapter 11). These are background rates for a warm aqueous cell, not constants of one experiment.

Ultraviolet light joins adjacent pyrimidines into cyclobutane dimers, mostly thymine dimers, plus a smaller yield of 6–4 photoproducts. The dimer kinks the helix and blocks a replicative polymerase. Oxidised bases such as 8-oxoguanine mispair with adenine. They do not give sources, doses, or procedures.

Mismatches are polymerase errors that escaped proofreading, including small loops from slippage on repeats. Double-strand breaks are a separate class: both strands are interrupted, so no intact complementary strand remains opposite the lesion. A nick-repair pathway cannot silently close a break.

5.2 Mismatch repair #

The study of Mismatch repair provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.

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5.2.1 MutS, MutL, and the choice of strand #

Both bases in a mismatch are normal DNA, so the pathway must decide which strand is wrong. The wrong strand is the new one.

In E. coli, Dam methylase modifies the adenine in GATC. Once Dam has caught up, this particular clock stops. It is not the human mechanism.

Eukaryotes have MutS and MutL homologues and no MutH. MutSα (MSH2 with MSH6) recognises base mismatches and very small loops. MutSβ (MSH2 with MSH3) recognises longer loops. MutLα (MLH1 with PMS2) cuts the new strand.

Germ-line loss of MLH1, MSH2, MSH6, or PMS2 causes Lynch syndrome. The tumours show microsatellite instability, because slippage loops are exactly what this pathway removes.

5.3 Excision of damaged bases #

5.3.1 Base-excision repair #

Base-excision repair removes a small lesion that does not strongly distort the helix. A glycosylase cuts the glycosidic bond and leaves an AP site. Other glycosylases recognise 8-oxoguanine or particular alkylated bases.

An AP endonuclease cuts the backbone. In the short-patch route, DNA polymerase β inserts one nucleotide and ligase seals the nick. In the long-patch route, pol δ or pol ε, with PCNA, inserts several nucleotides and displaces a flap, which FEN1 removes. The patch is a few nucleotides, not a long oligonucleotide.

5.3.2 Nucleotide-excision repair and xeroderma pigmentosum #

Nucleotide-excision repair looks for a bulky distortion, not for one wrong base. A thymine dimer or a large adduct is cut out inside an oligonucleotide, and the gap is filled.

In bacteria, UvrA with UvrB finds the distortion. UvrB verifies it and UvrA leaves. UvrC cuts on both sides of the lesion, releasing an oligonucleotide of about twelve nucleotides, and the helicase UvrD strips that piece out. DNA pol I fills the gap and ligase seals it. UvrD is not a fourth recognition subunit.

Eukaryotes use a larger set, many of the proteins named XP from xeroderma pigmentosum. In global repair, XPC finds distortions anywhere. TFIIH, met again in Chapter 6, opens the helix: its XPB and XPD subunits are the helicases. XPF–ERCC1 and XPG cut on either side and release about 24–32 nucleotides.

Classical xeroderma pigmentosum is a defect in this excision. Sun-exposed skin is extremely sensitive, and skin cancers are very frequent, because dimers are not cleared. The variant form is not an excision defect. It is a failure of DNA polymerase η, which normally copies past a thymine dimer by inserting adenines. Cockayne syndrome, concentrated in the transcription-coupled branch, is mainly a developmental and degenerative disease rather than a cancer of exposed skin. Losing the genome-wide pathway and losing the transcription-coupled pathway are not the same illness.

5.4 Double-strand breaks #

5.4.1 Homologous recombination and non-homologous end joining #

Non-homologous end joining recognises broken ends with Ku70–Ku80, and in vertebrates often the kinase DNA-PKcs. Ends are brought together, trimmed or filled if they are ragged, and sealed by DNA ligase IV with XRCC4. The junction often gains or loses a few nucleotides, because no template is consulted. The pathway works through the cell cycle and is the usual choice in G1, when no sister chromatid exists.

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Homologous recombination uses a sister chromatid, or less safely the homologous chromosome, as donor. The 5′ ends are resected to leave 3′ overhangs, which a recombinase filament uses to invade the donor duplex. Synthesis then extends from that 3′ end. Repair is usually accurate when the sister is the donor, because the sister is the copy just made. Use of the other parental chromosome can produce a crossover. Genetics owns what that exchange does to inheritance.

The sister exists only after replication, so the choice tracks the cell cycle. Cyclins do not perform the repair.

5.4.2 RecA and Rad51 #

RecA in bacteria and Rad51 in eukaryotes are the recombinases. With ATP they form a helical filament on single-stranded DNA. BRCA2 and other mediators load Rad51 and hand the DNA over from RPA. A filament does not form properly if RPA will not yield. Loss of BRCA2 impairs homologous recombination and predisposes to breast and ovarian cancer. That is evidence about the pathway, not a description of a treatment.

In E. coli a RecA filament on single-stranded DNA also helps LexA cleave itself, which induces the SOS genes, including error-prone polymerases. That regulatory response is bacterial. It is not a eukaryotic pathway.

Rad51 is the mitotic recombinase. The meiotic paralogue is Dmc1, and Spo11 makes the programmed breaks of meiosis. Meiotic chromosome behaviour is genetics and cell biology.

5.5 Key terms, summary, and self-check #

Key terms. AP site; thymine dimer; MutS; Dam; glycosylase; UvrABC; xeroderma pigmentosum; NHEJ; Ku; Rad51; BRCA2.

Summary. Uracil and abasic sites are removed by base excision of one or a few nucleotides. Bulky lesions such as thymine dimers are cut out as an oligonucleotide; classical xeroderma pigmentosum is the human defect in that pathway. Mismatches are excised from the new strand, marked in E. coli by a transient lack of Dam methylation and in eukaryotes by replication nicks and clamp orientation. Double-strand breaks are joined by Ku and ligase IV, often with a small change at the junction, or rebuilt by homologous recombination, in which a RecA or Rad51 filament on a 3′ overhang invades a donor. The accurate donor is the sister chromatid, and it exists only after replication.

Self-check.

1. Which spontaneous reaction produces uracil, which produces an AP site, and which enzyme does base excision use only for the first?

2. How does E. coli choose the strand to excise in mismatch repair, and why is Dam methylation the wrong answer in a human cell?

3. What does UvrABC recognise that a glycosylase does not, and which disease is a defect of the eukaryotic version?

4. Why is non-homologous end joining the usual choice in G1, and what do Ku and ligase IV do that Rad51 does not?

5. What is the substrate of a Rad51 filament, and why does loss of BRCA2 impair homologous recombination?

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