Lecture #5

DNA Damage and mechanisms of repair

MBOC (old) Page 267-285 MBOC (new) Page 295-311

Concepts

•  Factors that can induce DNA Damage •  Describe repair mechanisms for the

following errors – Single nucleotide errors – Small regions of mismatch – Double strand breaks

DNA Replication has Proofreading mechanisms

•  DNA polymerase has two proofreading mechanisms –  Correct nucleotide has high affinity for the moving

polymerase –  Conformational change in DNA polymerase is required

after nucleotide binding but before covalent addition - check DNA strand

•  Exonucleolytic proof-reading activity –  Only adds to the 3� -OH of a primer strand –  Mismatched nucleotides do not make a correct base-

pair, thus disrupting the primer

DNA editing mode of DNA Polymerase

DNA editing mode of DNA pol

DNA polymerase

What happens in catastrophic cases of DNA damage?

•  If a cells DNA is heavily damaged the classic DNA repair pathways can not operate

•  Replicative DNA polymerase stalls when it encounters DNA damage in catastrophic emergencies –  less accurate back-up DNA polymerases are used –  Human cells have >10 such polymerases –  Some recognize specific types of DNA damage –  Others make �good guesses�

•  Lack exonuclease proof reading activity

Why is DNA repair so important?

What creates and/or propagates DNA damage?

•  UV radiation (sun burn) •  Ionizing radiation (X-ray machines) •  Chemicals (ENU)

•  Errors in DNA repair mechanisms

Above ground atomic test site - Nevada Source: US AEC 1955

Release of the radioactive isotope I131

Ken Case �Atomic cowboy�

"The greatest irony of our atmospheric nuclear testing program is that the only victims of United States nuclear arms since World War II have been our own people."

-Congressional investigation team member

Syndromes in Humans caused by defects in DNA repair machinery

Repairing a single nucleotide error

Some types of DNA damage occurs spontaneously in cells

•  DNA in human cells lose ~ 5,000 purine bases (A, G) every day – N-Glycosyl linkage to deoxyribose hydrolyses

•  DEPURINATION – Removal of amine groups from bases

•  DEAMINATION –  Every 100 bases per cell every day

Example of Depurination

Result is absent base but an intact sugar phosphate backbone

Example of Deamination

This hydrolysis occurs ~100 bases per day

Special circumstance of methylated C residues

~ 3% of C residues are methylated in vertebrates Deamination of C residues results in generation of �T�

Pyrimidine dimers

•  Usually occurs in cells exposed to UV irradiation

•  These form between two neighboring pyrimidines

•  The most common are thymidine dimers

Method by which chemical modifications produce mutations

Base Excision Repair

•  Uses enzymes called DNA glycosylases •  Recognizes altered bases in DNA and

catalyzes its removal from the sugar •  Recognition is based upon a �flipping-out�

mechanism •  The catalyzed base is recognized by an AP-

endonuclease •  DNA polymerase repairs the gap

DNA Glycosylases detect �flipping out�

Base excision repair

Removing more than one base from a single strand

Nucleotide Excision Repair

•  Removes large changes in structure of DNA •  Multienzyme complex scans DNA for

distortions •  Cleaves the DNA strand, requires the activity

of DNA polymerase and Ligase to cooperate in DNA repair

Example - Pyrimidine dimers

Covalent bonds bind pyrimidines together C or T

Nucleotide Excision Repair

Transcription-Coupled Repair

•  Genomic DNA is constantly under surveillance •  DNA polymerase has inbuilt repair mechanisms

during replication •  RNA polymerase will also stall at genetic lesions

–  interact with coupling proteins –  recruits repair machinery to sites of mismatch

•  This is important for genes being expressed at the time of DNA damage

Cockayne�s Syndrome -  Homozygous recessive disorder -  Children age very quickly (this child is

8 years old) -  Growth retardation, skeletal

abnormalities, severe sensitivity to sunlight

-  Syndrome is believed to perpetuate because RNA polymerase becomes stalled at sites of DNA damage in important genes

-  As a result, important genes are not transcribed

Repair of Double Strand Breaks

Repair of Double Strand Breaks

•  This occurs when both DNA strands are cleaved •  Ionizing radiation (I131, replication errors, oxidizing

agents cell metabolites •  If not repaired, this can result in loss of

chromosomal regions and genes which are not segregated in mitosis

•  Two major mechanisms exist to repair double strand breaks –  Non-homologous end joining –  Homologous recombination

Non-Homologous End joining

Non-Homologous end joining

�Quick and Dirty��

NHEJ is a common repair mechanism used by cells

DNA Ligase IV

•  Acts specifically with XRCC1 (X ray repair cross complementing protein) in non homologous end joining (NHEJ) to covalently link two strands of DNA

•  Involves two copies of XRCC1 and DNA ligase IV to bind to DNA with nicks or broken ends

Homologous Recombination

DNA Damage and the Cell Cycle

BLOCK

BLOCK

SLOW

Rapid localization of repair proteins to double strand breaks

DAPI

BrdU

Mre11

Summary

•  Most damage to DNA can be repaired by one of two major DNA repair pathways

•  Double strand breaks pose a larger risk to the cell, and dire consequences for a cell�s biology including predisposition to cancer

•  Double strand breaks are repaired by a quick and dirty non homologous end joining method or by homologous recombination

Summary

•  DNA repair is a fundamental process required in all cells

•  Basic mechanisms to repair nucleotide errors utilize DNA glycosylases, DNA polymerase, DNA ligase

•  Double strand breaks are repaired by non-

homologous end joining or by homologous recombination