7/31/01

Lecture #13 - Mutagenesis, DNA repair - part I

DNA Damage

1) Replication errors - rare, in DNAP III only 1/10⁹

2) Chemical

a) Internal chemical breakdown largest nuber of overall errors - due to chemical environment of cell, e.g. acids, free radicals, etc.)

b) External chemicals - small number of overall errors, BUT, cells don't have DNA repair mechanisms for this damage

Types of Lesions

1) Single base changes

transition: G-C <--> A-T

purine <--> purine, pyrimidine <--> pyrimidine

usually results in chemically similar a. a.

transversion: G-C <--> C-G; A-T <--> T-A

purine <--> pyrimidine or vise versa

transversions are more damaging because they produce chemically dissimilar amino acids

2) Larger scale defects

structural distortions, e.g. addition of bulky side group distorts helix

deletions/inversions

small, 1 or 2 bp, frameshift mutation may result

large, 10² - 10⁶ bp, may involve entire arms of chromosomes

Single base change mutations

A) Replication errors

• usually a transition mutation
• frequently due to keto-enol equilibrium
• Fig. 1.16
• even proofreading may miss this mistake in unlikely event that T still in enol form
• see handout

B) Deamination

• most often involves conversion C --> U
• Fig. 14.25
• chemical agents:
  • hydroxylamine (C --> U only)
  • nitrous acid
  • sodium bisulfite
• may also occur spontaneously
• Types:
  • C --> U
  • A --> hypoxanthine (abnormal purine)
  • Hypoxanthine may base pair with C
• see handout

C) Base analogs

• most often an artificial situation used in lab
• base analogs are incorporated into DNA when the DNA is replicated
• BrdU: 5 bromouracil
  • pairs with A in keto form
  • pairs with G in enol form
  • Br side group shifts equilibrium towards enol form
  • causes more frequent mutations
• Fig. 1.16: enol form base pairs with G

Structural Distortions

A) Double stranded breaks

• done with X-rays, g-rays
• fragments DNA into pieces
• lethal for a haploid organism because it can't be repaired
• not so bad for diploids since can use undamaged copy as as template to repair damage

B) Pyrimidine/ Thymine Dimers

• cause by UV-light
• forms pyrimidine (thymine) dimers, also called cylcobutyl dimers
• 2 adjacent T bases form a ring, covalently bound
• ring structure distorts DNA
• replication and transcription machinery are blocked and can't read through
• Fig. 14.26

C) Intercalating Agents

• ethidium bromide, acridine (see handout for acridine structure)
• mechanism:
• agents noncovalently insert themselves between the base pairs
• "tricks" DNA replication machinery into thinking that the intercalating agent is a nucleotide in DNA
• RESULT: extra nucleotide inserted
• If this happens in an ORF (open-reading frame) may be disrupted causing a frameshift mutation
• see handout

D) Alkylation

• addition of methyl or other alkyl groups on the the DNA bases
• results in structural distortions
• may alter base pairing characteristics of bases
• e.g. addition of methyl group to O-6 of guanine
• chemical mutagens:
  • EMS: ethylmethanesulfonate
  • nitrosoguanidine
• Fig. 14.26
• note: not all methylation of DNA is damaging

E) Depurination

• removal of the base (purine) but the phosphate-sugar backbone remains intact
• largely spontaneous
• ß-N-glycosidic bond (bond that connects sugar and base) is hydrolyzed, releasing base
• abasic site results: DNA backbone sans base
• treating with strong acid promotes depurination

Environmental mutagens

• see handout
• benzo[a]pyrene - source: smoke - effects: intercalates in DNA, cause frameshifts
• nitrous acid - source: some prepared foods - effects: deaminates C --> U, leads to missense
• dimethyl nitrosamine - source: some prepared foods - effects: methylating agent, modifies baes, yields missense
• aflatoxin - source: moldy nuts or grans - effects: alkylating agent, modifies guanine, causes missense
• UV-light - source: sunlight - effects: (+) sexy tan, (-) forms pyrimidine dimers

Extent of DNA Damage

staggering amount, even in the absence of environmental mutagens

1) Replication Errors

• Humans - error rate 1/10⁹
• genome 3 x 10⁹
• 3 errors / cell division
• in lifetime ~3 x 10¹⁶

2) Spontaneous Breakdown

• due to chemical nature of cells
• free radicals, acidic environments, reactive intermediates of metabolism
• each cell loses ~10,000 bases/day

Therefore DNA Repair Essential!!

DNA Repair

• elaborate mechanisms, organisms dedicate many resources, relatively universal
• may mechanisms of repair in E. coli resemble those in eukaryotic cells
• mutations in DNA repair machinery increase cancer rate

Direct Repair

• chemical reversal of DNA damage
• 2 examples:
  • 1) E. coli (not mammals)
    • photoreactivating enzyme (phr)
    • phr + 370 nm light --> phr* activated
    • removes thymine dimers by cleaving C-C bonds
  • 2) Direct repair of alkylation
    • specifically for methylation of the 0-6 of G bases
    • guanine methyltransferase
    • protein in E. coli and mammals
    • Cys at active site my bind Me group with high stability (not reversible)
    • "enzyme" can only be used once
    • thus, it is easy to saturate cells by adding lots of methylating agent, overwhelming their ability to repair damage

Excision Repair - general mechanism

• include BER, NER, and Mismatch repair
• general mechanism: Fig. 14.27
• DNA is damaged
• Endonuclease cleaves on both sides of damaged base
• Exonuclease/helicase removes several nts of DNA between nicks
• Polymerase synthesizes replacement DNA
• DNA ligase seals nicks

Base Excision Repair (BER)

• repairs deaminated, alkylated, etcŠ
• short patch
• 2 enzymes:
  • 1) glycosylase
    • cleaves ß-N-glycosidic bond of incorrect base
  • 2) AP endonuclease
    • cleaves single strand
• many different DNA glycosylases can remove a variety of damaged base (see handout)

Nucleotide Excision Repair (NER)

• also called very short patch
• look at E. coli but mammals also have NER machinery
• 4 factors:
• uvrA- ATPase, damage recognition (mainly thymine dimers, other structural distortions)
• uvrB - creates open-preinitiation complex, helicase activity
• uvrC - endonuclease, makes incision 7 nts 5' and 3-4 nts 3' of incorrect base, requires ATP
• uvrD - helicase, removes fragment
• Fig. 14.28

Mismatch Repair

• repairs DNA replication errors
• system for correcting based on the sequence of the template from newly replicated strand
• must be able to distinguish template form newly replicated strand
• methylations are at non-mutagenic positions
• dam methylase
  • restores fully methylated condition of hemimethylated DNA produced during replication
  • recognizes palindromic sequence
  • 5' GATC 3'
  • 3' CTAG 5'
  • dam methylase methylates N7 of A
• Fig. 13.24

Mismatch repair proteins

• MutS - recognizes mismatch mutation, binds to it
• MutL - two DNA-binding sites
• one binds to mismatch
  • one is used to translocate along DNA until a GATC seq. it encountered
  • hydrolysis of ATP drives this translocation
• MutH - endonuclease
  • cleaves unmethylated strand from GATC site to the mismatch site
• MutU - helicase
  • helps release fragment
• mechanism: Figs.14.29, 14.30, handout
• mismatch correction system recognizes unmodified GATC sequence and mismatched base in the same DNA strand
• repair system removes DNA including mismatched pair from the strand containing unmethylated GATC
• DNAP fills the gap, replacing the mismatched base with correct base

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