# HELICASE CATALYZED DNA UNWINDING

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $511,032

## Abstract

Abstract
DNA helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single
stranded (ss) DNA intermediates required for 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. Despite extensive study, the mechanism of RecBCD-catalyzed DNA unwinding is not
understood. There is also little known about how the two motors communicate within RecBCD and the
allosteric regulation of its motor and nuclease activities by "chi". We have developed novel ensemble
fluorescence assays that enable us to monitor ssDNA translocation of the two motors independently.
This led to our discovery 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. We also recently discovered that RecBCD can unwind duplex DNA processively
even in the absence of ssDNA translocation by the canonical RecB and RecD motors indicating that
DNA melting and ssDNA translocation are separate processes. We have identified two domains within
the enzyme that enable such activity. Our goals are to: 1- understand the mechanism by which RecBC
and RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by its
canonical motors, 2- understand the allosteric regulation of DNA unwinding and ssDNA translocation
by the RecB nuclease domain and "chi", and 3- probe the conformational changes/domain movements
within RecBCD and RecBCD-DNA complexes that occur during its activities. 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 this complex multi-motor
enzyme translocates along and unwinds DNA and is regulated. Such studies will provide new insight
into nucleic acid motor enzymes that are essential for the maintenance of all genomes.

## Key facts

- **NIH application ID:** 9828720
- **Project number:** 5R01GM045948-28
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Timothy M Lohman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $511,032
- **Award type:** 5
- **Project period:** 1991-08-01 → 2020-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9828720

## Citation

> US National Institutes of Health, RePORTER application 9828720, HELICASE CATALYZED DNA UNWINDING (5R01GM045948-28). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9828720. Licensed CC0.

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