DNA helicases are ATP-dependent molecular motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA metabolism and genome maintenance in all organisms. Defects in DNA helicases are responsible for a number of human diseases. We are studying the mechanisms of DNA unwinding and ssDNA translocation of a multi-subunit DNA helicase/nuclease, E. coli RecBCD, which functions in repair of DNA double strand breaks and recombination. RecBCD is a hetero-trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (RecB, a 3'to 5'motor and RecD, a 5'to 3'motor) that move with different rates while part of the same complex, but undergo a switch in relative rates after the RecC subunit recognizes an 8 nucleotide ssDNA sequence, called "chi". The nuclease activity of RecBCD is also changed dramatically due to an allosteric effect of chi recognition. Although much is known about helicases, the basic mechanism(s) of DNA unwinding is still poorly understood. There is also little known about how the two motors might communicate within a helicase like RecBCD. We have developed a novel fluorescence assay that enabled us to discover that, in addition to its primary 3'to 5'translocase, RecBC (without RecD) also possesses a previously unrecognized secondary translocase activity that moves RecBC along the opposite DNA strand. Hence, the one RecB motor drives two translocase activities. Our goals are to: 1- determine the location of the secondary RecBC translocase within RecBC and if it functions within RecBCD;2- use our novel fluorescence assay to determine whether the RecB and RecD motors communicate during DNA unwinding by RecBCD and test our hypothesis that the secondary translocase activity plays a functional role in the communication between the two motor subunits (RecB and RecD) and their regulation by chi;3- determine the mechanism by which RecBCD and RecBC can melt out 4-6 bp from a blunt ended DNA in a Mg2+- dependent, but ATP-independent process and, 4- determine if DNA melting occurs separately from ssDNA translocation during DNA helicase activity. Thermodynamic, transient kinetic, structural and single molecule approaches (fluorescence and optical tweezers) will be used to obtain a molecular understanding of the kinetic mechanism(s) by which these molecular motors translocate along and unwind DNA and of how this multi-component helicase is regulated. Such studies will provide new insight into these nucleic acid motor enzymes that are essential for the maintenance of all genomes.

Public Health Relevance

DNA helicases and translocases play fundamental roles in all aspects of DNA metabolism, including DNA replication, recombination and repair in all organisms including humans. Mutations in a number of human DNA helicases are linked to several human diseases, including Werner's and Bloom's syndromes. Because of their pivotal roles in nucleic acid metabolism in all organisms, including bacteria, these enzymes are prime targets for drugs or antibiotics that may inhibit them specifically, and it is therefore important o understand their mechanisms of action.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045948-22
Application #
8459976
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
1991-08-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
22
Fiscal Year
2013
Total Cost
$478,443
Indirect Cost
$163,678
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Simon, Michael J; Sokoloski, Joshua E; Hao, Linxuan et al. (2016) Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation. J Mol Biol 428:2997-3012
Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto et al. (2016) Chemo-mechanical pushing of proteins along single-stranded DNA. Proc Natl Acad Sci U S A 113:6194-9
Petrova, Vessela; Chen, Stefanie H; Molzberger, Eileen T et al. (2015) Active displacement of RecA filaments by UvrD translocase activity. Nucleic Acids Res 43:4133-49
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Qiu, Yupeng; Antony, Edwin; Doganay, Sultan et al. (2013) Srs2 prevents Rad51 filament formation by repetitive motion on DNA. Nat Commun 4:2281
Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M (2012) SSB-DNA binding monitored by fluorescence intensity and anisotropy. Methods Mol Biol 922:55-83
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Antony, Edwin; Kozlov, Alexander G; Nguyen, Binh et al. (2012) Plasmodium falciparum SSB tetramer binds single-stranded DNA only in a fully wrapped mode. J Mol Biol 420:284-95

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