skip to main content


Title: The Escherichia coli clamp loader rapidly remodels SSB on DNA to load clamps
Abstract

Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading.

 
more » « less
Award ID(s):
1817869
PAR ID:
10385240
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Nucleic Acids Research
Volume:
50
Issue:
22
ISSN:
0305-1048
Page Range / eLocation ID:
p. 12872-12884
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

     
    more » « less
  2. null (Ed.)
    Abstract Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations. 
    more » « less
  3. Abstract

    Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32–ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32–ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.

     
    more » « less
  4. Redβ is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redβ is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redβ can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redβ with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redβ and SSB involves the C-terminal domain (CTD) of Redβ and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redβ CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redβ and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redβ-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology. 
    more » « less
  5. Abstract

    In Escherichia coli, the single-stranded DNA-binding protein (SSB) acts as a genome maintenance organizational hub by interacting with multiple DNA metabolism proteins. Many SSB-interacting proteins (SIPs) form complexes with SSB by docking onto its carboxy-terminal tip (SSB-Ct). An alternative interaction mode in which SIPs bind to PxxP motifs within an intrinsically-disordered linker (IDL) in SSB has been proposed for the RecG DNA helicase and other SIPs. Here, RecG binding to SSB and SSB peptides was measured in vitro and the RecG/SSB interface was identified. The results show that RecG binds directly and specifically to the SSB-Ct, and not the IDL, through an evolutionarily conserved binding site in the RecG helicase domain. Mutations that block RecG binding to SSB sensitize E. coli to DNA damaging agents and induce the SOS DNA-damage response, indicating formation of the RecG/SSB complex is important in vivo. The broader role of the SSB IDL is also investigated. E. coli ssb mutant strains encoding SSB IDL deletion variants lacking all PxxP motifs retain wildtype growth and DNA repair properties, demonstrating that the SSB PxxP motifs are not major contributors to SSB cellular functions.

     
    more » « less