Characterisation of interaction between hSSB1 and MRN complex in homologous recombination DSB repair pathway

   Homologous Recombination Non Homologous End Joining 2. NIJMEJEN BREAKAGE SYNDROMME DSB are repaired using 2 pathways 1.  NHEJ is a highly error prone repair pathway and it includes just joining of broken DNA ends and can lead to mutations and loss of DNA .INTRODUCTION  Double-strand breaks (DSBs) ±Highly geno toxic lesions chromosome breakage and rearrangement. mutagenesis and loss of crucial genetic information and cancer Related diseases ATM. ATLD.

HOMOLOGOUS RECOMBINATION HR error free Double strand break repair pathway MRN complex initiate the pathway  Localise to site of damage and initiate a 5¶ to 3¶ resection Activates ATM a key phosphokinase ATM is key for downstream regulation of proteins involved in the pathway Which include Rad51. BRCA2-crucial for strand invasion Fig1:Homologous Recombination pathway .

Rad50 and Nbs1. Initiates the Damage response by forming nuclear foci Binds to DNA DSB with DNA binding motifs of MRE11 and forms a globular domain along  with Rad50 Walker A & B motif Bridging of DNA molecule is driven by cxxc motif which dimerize in coordination with ZN 2+ ion NBS1 is identified to play a key role in the recruitment of the complex to the site of damage MRN complex is shown to have a strong 3¶ to 5¶ exonuclease activity .MRN COMPLEX MRN complex-Mre11.

phosphorylation CRUCIAL FINDINGS IN OUR LAB (unpublished work-data not shown) hSSB1 is crucial for the localisation of the MRN complex to site of damage Hssb1 essential for the 5¶ to 3¶ activity exonuclease activity of MRN complex for the processing of Double strand DNA breaks .e.Human Single strand DNA binding Protein (hSSB1) A novel protein and human analogue of archeal SSB key player in IR induced DNA double strand repair  crucial for activation of ATM Observed to facilitate binding of RAD51 to DSB site Bind to DNA independent of ATP i.

PROJECT OUTLINE INVITRO EXPRESSION AND PURIFICATION OF MRN COMPLEX 1. Isolated using Ni NTA Beads specific for His tagged hSSB1 3. Isolated by making use of Flag tagged Mre11 and His Tagged RAd50 3. Crosslinked with Cyanogen Bromide beads . Purified using FPLC techniques hSSB1 and 3 of its mutants( expressed in Bacteria) 1. Hssb1 Short. Complex expressed with the help of Baculovirus system in insect cells 2. hSSb1 shorter 2. hSSB1 wild hSSB1 T117E mutant( phospho mimic).

No known Motifs 3. RAD50B. Mre11B-DNA binding A domain 3. 2. Rad50A-Walker A and part of CxxC domain 2. NBS1c-Mre11 binding motif . NBS1b. Rad50A-Walker b and part of CxxC domain NBS1 1. Mre11A-Phosphoesterase domain.CxxC domain and Cxxc motif 3. NBS1A-FHA and BRCT motif 2. RAD50 and Nbs1 protein fragments Proteins broken into 3 fragments of almost equal sizes and tagged with GST MRE11 1. Mre11C-GAR motif and DNA binding B motif RAD50 1.INVITRO EXPRESSION OF INDIVIDUAL Mre11.

MRN complex interaction with hSSb1 and its Mutants .

hSSB1 & Mutants with MRE11A.MRE11B and MRE11C .

hSSB1 & mutants with Rad50A. Rad50B and RAD50C .

Nbs1C .hSSB1 & Mutants with Nbs1A. Nbs1B .

SIGNIFICANCE .

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