# Mechanisms of Helicases, Translocases and SSB Proteins involved in Genome Maintenance

> **NIH NIH R35** · WASHINGTON UNIVERSITY · 2020 · $527,645

## Abstract

Abstract
We are studying two classes of DNA binding proteins, DNA helicases and single stranded (ss)DNA
binding (SSB) proteins, that are both essential for genome maintenance in all organisms. DNA
helicases are ATP-dependent molecular motors that unwind duplex DNA to form the single stranded
(ss) DNA intermediates required for DNA replication, recombination and repair. SSB proteins bind
tightly to these ssDNA intermediates, protecting the DNA, but also bind directly to at least 17 other SSB
interacting proteins (SIPs) to bring them to their sites of action. Defects in DNA helicases are
responsible for a number of human diseases. We are undertaking quantitative studies of 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, as well as the
E. coli UvrD and Rep helicases which function in several DNA repair pathways. 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). Despite extensive study, the mechanism of helicase 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 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. This ability is regulated by its nuclease and arm domains. We are studying UvrD and Rep to
address the question of what is needed to turn a ssDNA translocase into a helicase and how this is
regulated. By accessory proteins such as MutL and PriC. Activation of a helicase is not well understood
process. E. coli SSB protein is a central player in all aspects of DNA metabolism. It can bind ssDNA in
multiple binding modes that differ dramatically in their properties, in particular ssDNA binding
cooperativity. A major focus is on the four intrinsically disordered C-terminal tails of SSB that we have
shown regulate cooperative binding of SSB to ssDNA and control the binding of the 17 SIPs. We have
developed SSB variants that selectively stabilize the different SSB binding modes and that have
different numbers of C-terminal tails and different properties and will determine how these affect protein
binding and DNA replication. An array of approaches, including thermodynamic, transient kinetic,
structural and single molecule approaches (fluorescence and optical tweezers), will be used in these
studies.

## Key facts

- **NIH application ID:** 9936743
- **Project number:** 1R35GM136632-01
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Timothy M Lohman
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $527,645
- **Award type:** 1
- **Project period:** 2020-05-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9936743

## Citation

> US National Institutes of Health, RePORTER application 9936743, Mechanisms of Helicases, Translocases and SSB Proteins involved in Genome Maintenance (1R35GM136632-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9936743. Licensed CC0.

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