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UID:941@biology.technion.ac.il

DTSTART;TZID=Asia/Jerusalem:20210120T130000

DTEND;TZID=Asia/Jerusalem:20210120T140000

DTSTAMP:20210802T125903Z

URL:https://biology.technion.ac.il/en/seminars/ph-d-graduate-seminar-sivas
 ubramanya-mangapuram-2/

SUMMARY:Ph.D. Graduate Seminar- Sivasubramanya Mangapuram [No Categories]
DESCRIPTION:Location:   \n Affiliation: \n Host:\n Characterizing the auxil
 iarynucleotide binding sites in RecBCDDNA helicase\, towards deciphering t
 heir functional roleAbstract:RecBCD is aDNA helicase-nuclease\, powering t
 he initiation of dsDNA break repair. It is ahighly processive fast helicas
 e\, with unwinding rates approaching 1\,600 bp/s.Yet\, there is no underly
 ing biophysical model to understand this fast-unwindingvelocity. To unrave
 l RecBCD'smechanochemical basis for its robust performance on DNA\, we hav
 e employedquantitative biophysical studies utilizing rapid kinetics\, ther
 modynamics\,cross-linking mass spectrometry (CLMS) and in-vivosurvival ass
 ays. Previous work in our lab had demonstrated the existence ofauxiliary b
 inding sites in RecBCD\, specifically in the RecCsubunit\, where ATP binds
  with lower affinity and with distinct chemicalinteractions as compared to
  the known catalytic sites. According to our model\,at intermediate ATP co
 ncentrations\, RecBCD achieves its fast-unwinding rate byutilizing the aux
 iliary binding sites to increase the flux of ATP to itscatalytic sites. Th
 e number of nucleotide-binding sites determined byequilibrium dialysis has
  been estimated to be at least four. While RecB and RecD\,each have one kn
 own structurally and chemically well-defined nucleotide-bindingsite\, the 
 additional two (or more) auxiliary nucleotide-binding sites remainedunknow
 n. Our current study strongly supports that they are likely to be locatedi
 n RecC. Inthis work\, we have attempted to abolish the nucleotide-binding 
 sites in RecC byusing a multi-step approach. A list of possible nucleotide
 -binding sites wasobtained by using a combination of CLMS and molecular do
 cking studies. Based onthis\, several mutations in RecC were carefully des
 igned and successfullycloned and purified to produce a variety of RecBCmut
 Ds. RecBCmutD proteins have an activated DNA ATPaseactivity\, with a highe
 r KM\,ATP.Furthermore\, equilibrium nucleotide binding\, DNA unwinding\, a
 nd invivosurvival assays suggest that the RecBCmutDs donot display the sam
 e biochemical behavior as the WT RecBCD. Inaddition\, despite exhibiting r
 apid transition\, as suggested by the rate ofunwinding performed by RecBCD
 \, wecan still observe a stepping mechanism essential to translocate along
  its DNAtracks. Our work sheds light on a novel and fundamental enzymatic 
 mechanismexhibited by a molecular motor.Beyond the mechanism of RecBCD and
 how it is evolved for its cellular function\, the question of why such an 
 enzymeto repair dsDNA break in humans did not evolve\, continues to be per
 tinent.  

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