The annealing helicase and branch migration activities of Drosophila HARP.

George A Kassavetis,James T Kadonaga,Sue Cotterill

doi:10.1371/journal.pone.0098173

George A Kassavetis, James T Kadonaga + Show 1 more

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https://doi.org/10.1371/journal.pone.0098173

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Journal: PloS one	Publication Date: May 27, 2014
Citations: 36	License type: CC BY 4.0

Affiliation: University of California, San Diego

Abstract

HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.

Highlights

HARP (hepA-related protein; called SMARCAL1 in Homo sapiens and MARCAL1 in Drosophila melanogaster) is a distant member of the SNF2 family of helicase-like ATPases
HARP is categorized as an a hydrolysable NTP (ATP)-dependent annealing helicase based on its ability to rewind complimentary single stranded DNA that is otherwise stably maintained by the ssDNA-binding protein, Replication Protein A (RPA) [3]. hsHARP does not bind stably to ssDNA or fully double stranded DNA, but does bind with high affinity to a number of DNA structures, including DNA forks, ssDNA:dsDNA junctions with extended 59or 39-ssDNA overhangs, heteroduplex DNA bubbles, internal ssDNA gaps and Holliday junctions
We examined the ability of dmHARP, which naturally contains only one HARP domain, to branch migrate DNA structures that were previously shown to serve as substrates for hsHARP

Summary

Introduction

HARP (hepA-related protein; called SMARCAL1 in Homo sapiens (hs) and MARCAL1 in Drosophila melanogaster (dm)) is a distant member of the SNF2 family of helicase-like ATPases. We examined the ability of hsHARP and dmHARP to branch migrate bidirectional replication bubbles, replication bubbles that contain an RNA:DNA hybrid, and a replication fork containing a highly stable poly rG:dC hybrid. We found hsHARP and dmHARP displayed comparable annealing helicase, D-loop disruption, branch migration of Holliday junctions, and branch migration of DNA and RNA:DNA hybrid-containing bidirectional replication bubbles, activities.

Results

Conclusion