Genetic stability of organisms

Accurate DNA replication+

DNA repair mechanismsPresent in prokaryotes and eukaryotes

1 million individual lesions/cell/day• Heat• Metabolic accident• Radiation• Environment• depurination

Molecular Biology of the Cell

But only 1/1000 accidental base change results in a permanent

mutation

Danger of DNA damage• Structural damage -> prevent replication/transcription

– Stalling of replication fork • Harmful mutations -> impair survival of organism

– Mutation in tumour suppressor genes for examples

• If unrepaired:– Senescence– Apoptosis– Aberrant cell division -> cancer

Genetic diseases associated with defects in DNA repair

2005 W. H. Freeman Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed.

ATM

Breast, ovarian and colon cancer

Defect in homologous recombination, BRCA2

Transcription-coupled repair

HNPCC

the DNA molecule can be repaired fairly easily because it carries 2 separate copies

of all the genetic information

Only viruses, that have a tiny genome (therefore tiny target for DNA damage) can afford to encode their genetic information in any molecule other than double stranded DNA single-stranded DNA or RNA.

Multiple DNA repair pathways

• Base excision repair (BER)• Nucleotide excision repair (NER)• Mismatch repair (MR)

• DNA strand cross link repair• Homologous recombination (HR)• Non-homologous end joining (NHEJ)

+ transcription coupled repair

Level of damage

Nucleotides known to be modified by:• Oxidative damage• hydrolytic attack• uncontrolled methylation

Purines (guanine and adenine) are more affected by those spontaneous reactions. 5000 purine bases are lost every day: DEPURINATION

Spontaneous DEAMINATION of cytosine uracil occurs at a rate of 100 bases per cell per day.

UV can covalently link two adjacent pyrimidine bases to form THYMINE DIMERS.

After T. Lindahl, Nature 362:709–715, 1993

Spontaneous DNA alteration

substitution deletion

Base excision repair Nucleotide excision repair

‘backup’ polymerases not as accurate as the normal replicative polymeraselack exonucleotic proofreading activity

2. Translesion DNA synthesis (TLS) polymerases

4. Reverse transcriptase Restrained to telomerase in eukaryotes, using a RNA template for DNA synthesis

1. Replicative polymerases

3. DNA repair and recombination

“Risky Business”

15 different mammalian DNA polymerases

Example of specialised polymerase: Terminal deoxynucleotidyl transferase (TDT) is expressed only in lymphoid tissue, and adds random nucleotides to double-strand breaks formed during somatic recombination to promote immunological diversity.

(post-replicative) DNA mismatch repaircorrects errors made by DNA polymerase during DNA replication

Defects in DNA mismatch repair have been found in several types of cancer, notably colon cancer, and microsatellite sequences that are either shorter or longer than normal are a hallmark of defective MMR.

MSH2 frequently mutated in hereditary nonpolyposis colon cancer (HNPCC)

In order to do this the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental)

(before sealing by DNA ligase)

Up to 1000 bp can be removed

DSBs are created biologically by the protein SPO-11 as thehighly regulated initiation of meiotic recombination.

Double stranded breaks

• Ionizing radiation• Replication errors• Oxidating agents

environment endogenous

Only used shortly after DNA replication, during interphase

Same genes, different alleles

Same genes, same alleles

Homologous recombinationHomologous recombination requires DSB of DNA (damage or stalled or broken replication fork), invasion of a homologous dsDNA molecule by a ssDNA end, pairing of homologous sequences, branch migration to form a Holliday junction, and isomerisation of the flanking sequences.

Important players

Rad51

BRCA1/2

FANCATM

Rad51, a sequence-independent DNA binding recombinase at the branch point

BRCA2 sequesters RAD51 via its BCR repeats and its C-terminal motif, mobilises it to the site of damage and then facilitates the formation of helical RAD51–single stranded DNA nucleoprotein filaments that search for a homologous DNA template.

Owen Richard Davies & Luca Pellegrini, Nature Structural & Molecular Biology 14, 475 - 483 (2007)

Holliday junction

ATM protein kinase• DSBs create changes in

chromatin structure which activate ATM by autophosphorylation, which then induces many cellular responses by phosphorylating a vast number of target proteins.

• Associate with the BRCA1-associated genome surveillance complex (BASC)

• Can phosphorylate p53 also involved in DNA repair and cell cycle arrest.

The Fanconi anaemia/BRCA pathway• The FANC protein family is involved in the recognition and repair of

damaged DNA. The FA complex is activated when DNA stops replicating because of damage. The core complex can associate with BRCA1 and BRCA2.

This complex mediates the monoubiquitylation of FANCD2. Activated FANCD2, in turn, is translocated to chromatin and DNA-repair foci. These foci contain the BRCA1 protein and is known to bind directly to RAD51 and to DNA, and to participate in homology-directed DNA repair.

BRCA1, in combination with BARD1, has E3 ubiquitin ligase activity; BRCA2 hasno enzymatic activity. Both BRCA1 and BRCA2 are tumour suppressor proteins.They form multiple protein complexes with overlapping functions.