DNA Damage and Repair

Sources of DNA Damage

Inherent instability of DNA in vivo

due to N-glycosidic bond – weakest bond in DNA; can also form enzymatically if base is damaged

deamination of C, A, G

deamination of C is greatest; amino side chains exposed to minor groove thus water; -C-NH₂ Þ -C=O

Physical attack

Chemical attack

deaminating agents – bisulfate

cross-linking agents – psoralen Þ bridges the 2 strands of DNA inhibiting replication

Response to DNA Damage

Apurinic (AP) nuclease pathway – missing base due to depurination (purine "falls off" DNA) or glycosylase enzyme

AP endonuclease recognizes strand with missing base, cuts away very small section of backbone, fills in with polymerase and ligase

Excision endonuclease pathway – distorted helix (abnormal bases, bulky adducts, etc)

Excision endonuclease removes distant bases on both sides of the error, then helicase removes the bases in btwn (~20 bases excised total), gapped DNA filled in with polymerase and ligase

Other important repair enzymes

Each enzyme recognizes a specific kind of damaged base
If no N-glycosylase corresponds to a type of damage, excision endonuclease is used
Some kinds of damage can be repaired by both

2 exceptions: reversing the damage

photoreactivation: breaks thymine dimers

Targeting of Repair Activity

Way to focus 1st on damage repair to actively transcribed seq; interaction btwn transcription complex and repair complex

Repair is restricted to damage in the non-template strand

Important factor is breast cancer: cells knocked out at BRCA1 gene are defective in transcription-coupled repair

Consequences of Unfixed Damage

p53 gene: delays replication to allow more time for DNA repair; greater the damage, greater the action; if DNA is heavily damaged, cell growth is permanently inhibited Þ cell death; mutated in some tumors

Repair of DNA replication errors: post-replication mismatch repair system

treats parental strand as correct and changes new strand
prokaryotes: strand discrimination relies on differential methylation of the strands
eukaryotes: strand discrimination is unknown
utilizes 2 proteins: Mut S (mismatch recognition protein); Mut L
(locates nick in new strand)
HNPCC: responsible for 15% of all colon cancers; loss of mismatch repair capacity

Clinical Impact of Damage and Repair

Xeroderma Pigmentosum

patients highly prone to develop skin cancer after exposure to sun light

Li-Fraumeni Syndrome