Structures and operating principles of the replisome

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Abstract

The replisome that performs concerted leading and lagging DNA strand synthesis at a replication fork has never been visualized in atomic detail. Using bacteriophage T7 as a model system, we determined cryo-EM structures up to 3.2 Å of helicase translocating along DNA, and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates hand-over-hand sequentially along a ssDNA coil, akin to the way AAA+ ATPases unfold peptides. Two lagging-strand polymerases are attached to the primase ready for Okazaki-fragment synthesis in tandem. A β-hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

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Maintaining genome stability at the replication fork.

Dana Branzei, Marco Foiani (2010)

Aberrant DNA replication is a major source of the mutations and chromosome rearrangements that are associated with pathological disorders. When replication is compromised, DNA becomes more prone to breakage. Secondary structures, highly transcribed DNA sequences and damaged DNA stall replication forks, which then require checkpoint factors and specialized enzymatic activities for their stabilization and subsequent advance. These mechanisms ensure that the local DNA damage response, which enables replication fork progression and DNA repair in S phase, is coupled with cell cycle transitions. The mechanisms that operate in eukaryotic cells to promote replication fork integrity and coordinate replication with other aspects of chromosome maintenance are becoming clear.

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Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.

Ivar Ilves, Tatjana Petojevic, James J Pesavento … (2010)

MCM2-7 proteins provide essential helicase functions in eukaryotes at chromosomal DNA replication forks. During the G1 phase of the cell cycle, they remain loaded on DNA but are inactive. We have used recombinant methods to show that the Drosophila MCM2-7 helicase is activated in complex with Cdc45 and the four GINS proteins (CMG complex). Biochemical activities of the MCM AAA+ motor are altered and enhanced through such associations: ATP hydrolysis rates are elevated by two orders of magnitude, helicase activity is robust on circular templates, and affinity for DNA substrates is improved. The GINS proteins contribute to DNA substrate affinity and bind specifically to the MCM4 subunit. All pairwise associations among GINS, MCMs, and Cdc45 were detected, but tight association takes place only in the CMG. The onset of DNA replication and unwinding may thus occur through allosteric changes in MCM2-7 affected by the association of these ancillary factors. Copyright 2010 Elsevier Inc. All rights reserved.

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Mechanism of DNA translocation in a replicative hexameric helicase.

Eric J. Enemark, Leemor Joshua-Tor (2006)

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.