Lecture 18 DNA Repair Rohini. K

Learning Objective 08 Nov 2011Rohini K FoM 2  An overview of the types of DNA damage  DNA repair mechanisms  Defects in DNA repair mechanisms.

Significance of mutation & repair mechanism 08 Nov 2011Rohini K FoM 3

1.Single base alteration 2.Two base alteration 3.Chain breaks Due to ionizing radiations 4.Cross linkages between bases and cross linkages between DNA and Protein Depurination,Deamination, Alkylation, Insertion, Deletion, Base substitution UV light induced Tymine- Thymine dimer 4 types of DNA Damage 08 Nov 2011Rohini K FoM 4

 Depurination– is removing of G or A from deoxyribose through hydrolysis of the N- glycosyl linkage.  Deamination of cytosine to uracil. [chemical change]  Thymine – dimers are formed by UV rays and ionizing radiation. Single Base Alteration 08 Nov 2011Rohini K FoM 5

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DNA REPAIR  Damage to the master molecule must be repaired or the organism’s very life is threatened.  The cell spends a lot of energy on enzymes that constantly monitor DNA for mutations.  The proof reading function of DNA polymerase II of E.coli identifies copying error and corrects them. 08 Nov 2011Rohini K FoM 9

DNA REPAIR----Cont  In DNA damage, only one of the 2 strands needs to be repaired.  Error in one strand can be repaired [bases removed and new ones added] by using the other strand as a template.  In this way, the correct bases are put in proper place to maintain a proper nucleotide sequence. 08 Nov 2011Rohini K FoM 10

REPAIR MECHANISM  The 4 basic steps in DNA repair are: (1) Recognize (2) Remove (3) Resynthesize (4) Religate --- Enzymes are very specific ---DNA Polymerase and ligase 08 Nov 2011Rohini K FoM 11

DNA REPAIR SYSTEMS  Base Excision Repair  Nucleotide Excision Repair  Mismatch Repair  Double Strand Break Repair 08 Nov 2011Rohini K FoM 12

BASE EXCISION REPAIR  The amino groups of cytosine adenine guanine [EX.] C to U Are susceptible to spontaneous deamination 08 Nov 2011Rohini K FoM 13

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Uracil DNA glycosylase recognizes the uracil and cleaves the N- glycosidic bond creating apyrimidinic site. [AP site] AP endonuclease nicks the phosphodiester backbone and excises the sugar-phosphate residue DNA polymerase replaces the correct base and ligase rejoins the DNA 08 Nov 2011Rohini K FoM 15

NUCLEOTIDE EXCISION REPAIR  replace damaged DNA up to 30 bases. [bulky lesion]  Recognizes damage by UV light damage [T-T dimers], smoking ionization radiation.  Once the lesion has been located an endonuclease activity UvrA, UvrB, UvrC cleaves the modified strand on both sides of the distortion and the entire lesion is removed. 08 Nov 2011Rohini K FoM 16

Nucleotide repair 08 Nov 2011Rohini K FoM 17

MISMATCH REPAIR  Faulty DNA replication mismatched base pair [Ex] C is copied opposite to A instead of T  Specific proteins MutS which complexes with Mut L, MutH scan daughter strand and pass the mismatched information to specific GATC endonucleases.  The parent template strand is methylated on GATC sequence  The daughter strand is initially unmethylated 08 Nov 2011Rohini K FoM 18

MISMATCH REPAIR--Cont  The differential methylation identify the strand with wrong nucleotide  The GATC endonuclease cuts the strand bearing the mutation.  An exonuclease then digests this strand from the GATC  This is followed by resynthesis and religation. 08 Nov 2011 Rohini K FoM 19

MISMATCH REPAIR DISORDER Hereditary nonpolyposis colorectal cancer (HNPCC)  The human homologues of MutS [hMSH2] and MutL [ hMLH1] have mutations  Also known as Lynch syndrome 08 Nov 2011Rohini K FoM 20

DOUBLE STRAND BREAK REPAIR  occurs due to ionization radiation or oxidative free radical ions.  Two proteins are involved---Ku and DNA-PK.  These proteins bind to the free ends of DNA.  This allows approximation of the separated ends DNA-PK phosphorylates Ku activates helicase, helps in unwinding.  The unwound DNA forms base pairs, the extra nucleotide tails are removed by exonuclease and the gaps are filled by ligase. 08 Nov 2011Rohini K FoM 21

DOUBLE STRAND BREAK REPAIR 08 Nov 2011Rohini K FoM 22

DISORDERS IN DNA REPAIR MECHANISM XERODERMA PIGMENTOSUM: Defective NER mechanism; sensitivity to UV light; skin cancer. BLOOM’S SYNDROME: Defect in DNA ligase or Helicase; lympho reticular malignancies. ATAXIA TELANGECTASIA : Defective NER mechanism; sensitivity to damage by UV and X-rays. HEREDITARY NONPOLYPOSIS COLON CANCER: Mismatch repair is defective; defect in hMSH1. 08 Nov 2011Rohini K FoM 23

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Learning Outcomes  Explain the various types of DNA damage.  Describe the various repair mechanisms to correct the DNA damage.  Explain the defects in DNA repair mechanism. 08 Nov 2011Rohini K FoM 25

References  Textbook of biochemistry: Lippincott’s Illustrated Reviews, 5th edition – Unit VI – Storage and Expression of Genetic Information. Chapter 29: DNA Structure, Replication & Repair  Textbook of biochemistry: Vasudevan 5 th Edition - Chapter 41 – DNA Structure and Replication. 08 Nov 2011Rohini K FoM 26

08 Nov 2011Rohini K FoM 27 Thank You