Abstract
The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.
Highlights
Gp59 is required to correctly load DNA helicase during T4 recombination-dependent DNA replication and repair transactions
These and other results indicate that Gp59 targets helicase assembly onto Gp32-single-stranded DNA (ssDNA) clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication
Evidence suggests that Gp59 from the helicase loading complex (HLC) is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks
Summary
Gp59 is required to correctly load DNA helicase during T4 recombination-dependent DNA replication and repair transactions. Gp32 and Gp59 are present on ssDNA in large, compact protein-DNA complexes that represent a major portion of the protein mass of the replication fork [19, 23] These results suggest that the co-localization of ssDNA with Gp59 and Gp32 is an important structural feature of T4 helicase/primosome assembly, forming an intermediate that we refer to as the helicase loading complex (HLC). Results show that HLC formation requires a minimum cluster size of Gp32 on ssDNA and that Gp59 preferentially co-localizes with Gp32-ssDNA clusters even in the presence of excess free ssDNA These and related findings support a model in which Gp59-Gp32 interactions promote helicase loading onto the displaced strand of D-loops, leading to efficient initiation of RDR and homology-directed repair. The results of this study provide new insights into genome stability mechanisms that are used by all organisms to repair DNA damage and avoid disease
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