Molecular Studies of Protein-DNA Interactions

Hsieh, P.

Abstract

We are examining the effects of mispaired bases on DNA branch migration. During homologous recombination, Holliday structures containing regions of heteroduplex DNA are formed. These heteroduplex regions can undergo branch migration which, in one direction, can increase the amount of genetic information that is exchanged between homologous chromosomes. Alternatively, net movement in the reverse direction will reduce the length of heteroduplex DNA. We are examining the role of E. coli recA protein in promoting branch migration through mispaired bases. In order to do so, we have initiated studies of nonenzymatic branch migration through mispaired bases. Using oligonucleotides to form three-stranded substrates, we observe that nonenzymatic branch migration does not proceed to any significant extent through as few as two mispaired bases (mismatches or deletions). An examination of branch migration in the presence of RecA nucleoprotein filaments formed in the presence of ATP reveals that RecA has only a very modest effect in promoting branch migration through mispaired bases. On the bases of these results, we suggest that recombinase proteins like RecA can accommodate mispaired bases in heteroduplex DNA by nucleating pairing of homologous DNAs in regions flanking mispaired bases. We are currently studying branch migration using four-stranded substrates. We are also initiating studies on the regulation of expression of the human ligase I gene. We have obtained cDNA probes corresponding to various regions of the ligase gene using Polymerase Chain Reaction (PCR) and are examining by Northern blot analysis whether the ligase gene is activated in response to UV damage in HeLa cells.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
• Type: Intramural Research (Z01)
• Project #: 1Z01DK052015-03
• Application #: 3855399
• Support Year: 3
• Fiscal Year: 1991

Institution

• Name: National Institute of Diabetes and Digestive and Kidney Diseases
• Country: United States

Publications

Li, Zhongdao; Pearlman, Alexander H; Hsieh, Peggy (2016) DNA mismatch repair and the DNA damage response. DNA Repair (Amst) 38:94-101

Yoshioka, Ken-ichi; Yoshioka, Yoshiko; Hsieh, Peggy (2006) ATR kinase activation mediated by MutSalpha and MutLalpha in response to cytotoxic O6-methylguanine adducts. Mol Cell 22:501-10

Yang, Yong; Sass, Lauryn E; Du, Chunwei et al. (2005) Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions. Nucleic Acids Res 33:4322-34

Schofield, Mark J; Hsieh, Peggy (2003) DNA mismatch repair: molecular mechanisms and biological function. Annu Rev Microbiol 57:579-608

Wang, Hong; Yang, Yong; Schofield, Mark J et al. (2003) DNA bending and unbending by MutS govern mismatch recognition and specificity. Proc Natl Acad Sci U S A 100:14822-7

Selmane, Tassadite; Schofield, Mark J; Nayak, Sunil et al. (2003) Formation of a DNA mismatch repair complex mediated by ATP. J Mol Biol 334:949-65

Biswas, I; Obmolova, G; Takahashi, M et al. (2001) Disruption of the helix-u-turn-helix motif of MutS protein: loss of subunit dimerization, mismatch binding and ATP hydrolysis. J Mol Biol 305:805-16

Hsieh, P (2001) Molecular mechanisms of DNA mismatch repair. Mutat Res 486:71-87

Schofield, M J; Nayak, S; Scott, T H et al. (2001) Interaction of Escherichia coli MutS and MutL at a DNA mismatch. J Biol Chem 276:28291-9

Junop, M S; Obmolova, G; Rausch, K et al. (2001) Composite active site of an ABC ATPase: MutS uses ATP to verify mismatch recognition and authorize DNA repair. Mol Cell 7:1-12