Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.

By Johannes C. Walter, J. Newport. 2000

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Subfields: Molecular Biology, Plant Science

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Continued primer synthesis at stalled replication forks contributes to checkpoint activation (2010, methodology): “7% Tris/borate/EDTA agarose gel containing 4 μM chloroquine (Sigma-Aldrich; Walter and Newport, 2000), and the gel was stained with SybrGold (Invitrogen).”
Treslin Collaborates with TopBP1 in Triggering the Initiation of DNA Replication (2010, background): “Mcm2 (a marker for the MCM complex) preceded Treslin in associating with chromatin , whereas Cdc45 commenced binding just after Treslin.”
Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. (2005, methodology): “The amount of DNA unwinding determines the level of Chk1 activation induced with aphidicolin We previously showed that replication of a 3-kb plasmid in the presence of aphidicolin results in extensive unwinding (Walter and Newport 2000).”
Mechanics of biomolecules (2002, background): “Similar to transcription, the initiation of replication also requires negative supercoiling of the DNA template (Walter and Newport, 2000)—a lack of unwinding prevents the DNA polymerase from binding.”
The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication (2002, methodology): “ORC depletion of NPE was performed as described (Walter and Newport, 2000).”
MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication (2017, background): “The association of Cdc45 with chromatin correlates closely with activation of the replicative helicase and initiation of replication in egg extracts (Mimura and Takisawa, 1998; Mimura et al., 2000; Walter and Newport, 2000).”
PARP is activated at stalled forks to mediate Mre11‐dependent replication restart and recombination (2009, background): “These ssDNA regions may represent the uncoupling of replicative polymerase and heli- case, which occurs at stalled forks and also generates ssDNA (Walter and Newport, 2000; Byun et al, 2005), these may be longer stretches of ssDNA, which we show do not activate PARP and may, therefore, be sensed in…”