DNA REPAIR

ELEMENT 3 LECTURE 18

LEARNING OBJECTIVES * Types of DNA Damage * Repair mechanism to correct the DNA damage and their defects

Significance of mutation & repair mechanism

1.Single base alteration 2.Two base alteration

Depurination,Deamination, Alkylation, Insertion, Deletion, Base substitution UV light induced Tymine- Thymine dimer

3.Chain breaks Due to ionizing radiations 4.Cross linkages between bases and cross linkages between DNA and Protein

4 types of DNA Damage

Depurination is removing of G or A from deoxyribose through hydrolysis of the N- glycosidic linkage between them Deamination of cytosine to uracil. [chemical change] --- not corrected C-G pair will be converted to A-T pair [mutation] Thymine dimers are formed by UV rays and ionizing radiation.

DNA REPAIR
Damage to the master molecule must be repaired or the organisms 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.

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.

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

DNA REPAIR SYSTEMS

Base Excision Repair Nucleotide Excision Repair Mismatch Repair Double Strand Break Repair

BASE EXCISION REPAIR The amino groups of cytosine Are susceptible to adenine spontaneous guanine deamination [EX.] C to U

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(cuts) the sugar-phosphate residue

DNA polymerase replaces the correct base and ligase rejoins the DNA

NUCLEOTIDE EXCISION REPAIR

replace damaged DNA up to 30 bases. [bulky lesion] Recognizes UV light damage [T-T dimers], damage caused by smoking, and damage caused by 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.

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.

NUCLEOTIDE REPAIR

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

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.

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.

DOUBLE STRAND BREAK REPAIR

DISORDERS IN DNA REPAIR MECHANISM XERODERMA PIGMENTOSUM: Defective NER mechanism; sensitivity to UV light; skin cancer. BLOOMS 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 and two genes

LEARNING OUTCOMES

Learning Outcomes

At the end of the lecture, you should be able to: * Explain the various types of DNA Damage * Describe the various repair mechanism to correct the DNA damage * Explain the defects in DNA repair mechanism

References
1. Harpers Illustrated Biochemistry 26th edn, Chapter 36: DNA organization, Replication and Repair 2. Lippincotts Illustrated Reviews Biochemistry 3rd ed, Chapter 29: DNA structure and Replication 3. Textbook of Biochemistry (Vasudevan and Sreekumari) 5th edn