The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA.

Dahai Gai,Damian Wang,Shu-Xing Li,Xiaojiang S Chen,Shu Xing Li

doi:10.7554/elife.18129

Abstract

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells.

Highlights

DNA replication is essential in the inheritance of genetic information for living organisms
Using electron microscopy (EM), we showed that a slightly longer LT108 construct can assemble as a double hexamer on dsDNA containing the full 64-bp core origin in the presence of ATP or ADP, and can support the replication of such DNA in HeLa cell extract (Cuesta et al, 2010)
These stable pre-formed hexamers/double hexamers on origin dsDNA are capable of unwinding the DNA when Mg2+ is supplied (Figure 1D,E), which is consistent with previous reports for full-length Large-T Antigen (LT) (Uhlmann-Schiffler et al, 2002)

Summary

Introduction

DNA replication is essential in the inheritance of genetic information for living organisms. For DNA replication in prokaryotic cells, the initiator protein, DnaA, first binds to the replication origin to initiate origin melting. The replicative DnaBfamily of hexameric helicases are loaded onto the fully melted single-stranded DNA (ssDNA) for replication fork unwinding [reviewed in O’Donnell et al (2013)] and references therein). For DNA replication in archaeal and eukaryotic cells, replicative helicases, such as minichromosome maintenance proteins (MCM) and SV40 Large T Antigen (LT), load onto the origin dsDNA as headhead (or N-N) double hexamers [reviewed in Fanning and Zhao (2009); Gai et al (2010); O’Donnell et al (2013); Slaymaker and Chen (2012) and references therein]. The origin bound by the double hexameric helicases, together with other replication factors, will eventually melt the duplex and produce a pair of replication forks. The molecular interactions of these hexamer helicases in archaeal and eukaryotic replication systems with their origin dsDNA have yet to be defined

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Results

Conclusion