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DNA-mediated charge transport in a biological context : cooperation among metalloproteins to find lesions in the genome

Citation

Sontz, Pamela Alisa (2012) DNA-mediated charge transport in a biological context : cooperation among metalloproteins to find lesions in the genome. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:05242012-110320685

Abstract

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. A molecular wire, DNA conducts charge with shallow distance dependence, yet mismatches and lesions attenuate this process. We have proposed a model where repair proteins, containing redox-active [4Fe4S] clusters, utilize DNA charge transport (CT) to scan the genome for lesions. Based on this model, proteins are predicted to redistribute onto strands where DNA CT is inhibited. Using single-molecule atomic force microscopy (AFM) we have probed the redistribution of EndoIII, a base excision repair protein that contains a [4Fe4S] cluster. Consistent with the model, we find a redistribution of EndoIII onto DNA strands (3.8 kbp) containing C:A mismatch, which is not a specific substrate of EndoIII but inhibits CT. Proteins with mutations making them deficient in DNA-mediated CT do not similarly redistribute onto mismatched strands.

Various DNA-binding proteins, such as those involved in repair and pathways that maintain the integrity of DNA, have been found to contain FeS domains and other redox cofactors. We are discovering proteins from alternate repair pathways that may also utilize DNA CT to find damage. XPD, a 5′-3′ helicase involved in nucleotide excision repair, contains a conserved [4Fe4S] cluster and exhibits a DNA-bound redox potential that indicates it is able to carry out DNA CT. In AFM studies, we observe also the redistribution of XPD onto strands containing a mismatch. We further demonstrate that an XPD mutant, L325V, defective in carrying out DNA CT, does not redistribute onto mismatched strands.

DNA CT between distinct repair proteins bound to DNA was also probed by AFM. When XPD and EndoIII are mixed together, they coordinate in relocalizing onto mismatched strands. However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs. These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between repair proteins in their search for damage in the genome.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Atomic Force Microscopy; Base Excision Repair; DNA-mediated Charge Transport; Repair Proteins; Iron-Sulfur Clusters
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Awards:The Herbert Newby McCoy Award, 2012
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Barton, Jacqueline K.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Clemons, William M.
  • Barton, Jacqueline K.
  • Tirrell, David A.
Defense Date:7 May 2012
Funders:
Funding AgencyGrant Number
National Institutes of HealthGM49216
NRSA Training Grant5T32GM07616
Record Number:CaltechTHESIS:05242012-110320685
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:05242012-110320685
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.ccr.2010.09.002DOIUNSPECIFIED
http://dx.doi.org/10.1073/pnas.0908059106 DOIUNSPECIFIED
http://dx.doi.org/10.1021/bi2003179DOIUNSPECIFIED
http://dx.doi.org/10.1073/pnas.1120063109DOIUNSPECIFIED
http://dx.doi.org/10.1021/ja205568rDOIUNSPECIFIED
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7072
Collection:CaltechTHESIS
Deposited By: Pamela Sontz
Deposited On:29 May 2012 21:41
Last Modified:26 Dec 2012 04:43

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