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Review
. 2014 Dec;71(23):4545-59.
doi: 10.1007/s00018-014-1721-1. Epub 2014 Sep 20.

Replication Initiation and Genome Instability: A Crossroads for DNA and RNA Synthesis

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Free PMC article
Review

Replication Initiation and Genome Instability: A Crossroads for DNA and RNA Synthesis

Jacqueline H Barlow et al. Cell Mol Life Sci. .
Free PMC article

Abstract

Nuclear DNA replication requires the concerted action of hundreds of proteins to efficiently unwind and duplicate the entire genome while also retaining epigenetic regulatory information. Initiation of DNA replication is tightly regulated, rapidly firing thousands of origins once the conditions to promote rapid and faithful replication are in place, and defects in replication initiation lead to proliferation defects, genome instability, and a range of developmental abnormalities. Interestingly, DNA replication in metazoans initiates in actively transcribed DNA, meaning that replication initiation occurs in DNA that is co-occupied with tens of thousands of poised and active RNA polymerase complexes. Active transcription can induce genome instability, particularly during DNA replication, as RNA polymerases can induce torsional stress, formation of secondary structures, and act as a physical barrier to other enzymes involved in DNA metabolism. Here we discuss the challenges facing mammalian DNA replication, their impact on genome instability, and the development of cancer.

Figures

Fig. 1
Fig. 1
Assembling an origin of DNA replication. Following DNA segregation in mitosis, the heterohexameric ORC complex associates with DNA. Cdc6 and Cdt1 associate with DNA-bound ORC, which facilitates the loading of the MCM2–7 replicative helicase and forms the pre-replicative complex (pre-RC). In response to growth signals, the cell approaches the decision point to enter into S phase and commit to DNA replication. Cyclin A-CDK2 activity marks entry into S phase, phosphorylating Cdc6 leading to its relocalization to the cytoplasm. Cyclin E-CDK2 recruits Cdc45 to the assembled pre-RCs, which in turn leads to Mcm10 association, which is required for MCM2–7 activation. The GINS complex then binds Cdc45 and MCM2–7 to form a complete replicative helicase. The regulatory proteins Treslin and TopBP1 associate with pre-RCs, which are now capable of initiating replication, forming pre-initiation (pre-IC) complexes. Entry into S phase is also characterized by the activation of the Cdc7-Dbf4 kinase, which phosphorylates and activates many proteins associated with the pre-IC, and is required for origin firing. Supercoiling unwinds DNA at the origin in a Cdc45-dependent manner, and leads to the loading of downstream replisome components, including the clamp loader RFC (blue circle) and its target PCNA (pink circle), which associates directly with the DNA polymerases. Pola primase and the leading and lagging strand polymerases Pole and Pold all load onto the open DNA behind MCM2–7, forming a functional replisome
Fig. 2
Fig. 2
Organization of DNA replication in metazoans. a Metazoan replication begins with origin firing in early replication zones, which are co-incident with actively transcribed euchromatic DNA and marks associated with active transcription. Late replicating zones, which associate with heterochromatic regions, also contain replication origins that fire later in S phase to complete synthesis in a timely manner. b Regulation of replication initiation in response to stress. In the absence of replication stress, replication initiates (green circle) from only one or a few widely spaced origins in early replication zones. Many origins do not fire (black circles, left). Late-replicating regions also contain origins (black circles, right), which fire later in S phase to complete replication in heterochromatic zones. In response to replication stress early in S phase, additional origins near the stress site (yellow star) also fire (orange circles), while origin firing in late-replicating DNA is suppressed. Origin firing near damage sites can help “rescue” stalled or collapsed replication forks by reinitiating replication on the opposite side of the lesion, completing replication within the damaged region (left). Suppression of late-firing origins may also suppress genome instability by preventing the initiation of replication in new genomic locations during potentially unfavorable conditions. Origins in distant regions will fire after the stress signal abates, following successful repair or adaptation after prolonged checkpoint activation
Fig. 3
Fig. 3
Histone modifications associated with both transcription and DNA replication/repair processes. A variety of epigenetic modifications link transcription with DNA replication and repair processes. A subset of proteins and histone modifications appear to play dual roles, and may allow for crosstalk in S phase. The promoters of active genes predominantly contain a nucleosome free region (NFR) 5’ of the transcription start site (TSS). Interestingly, the DNA repair proteins involved in transcription-coupled repair XPC, XPG and XPF have been found to play a role in regulating transcriptional activity at a subset of genes, and occupy promoters at the NFR, like many transcription factors (purple panel). Acetylation of histones H3 and H4 promotes transcriptional activity by enhancing chromatin accessibility and the adoption of a euchromatic state. H3/H4 acetylation also increases with homologous recombination (HR). Similar to its role in transcription, H3/H4 acetylation makes the DNA surrounding the DSB “accessible” for repair. HR requires the invasion of a homologous template (either the sister chromatid or homologous chromosome) to prime new DNA synthesis and repair the damaged region. Thus, “open” chromatin surrounding the DNA break and the repair template would increase repair efficiency (red panel). H3 methylation on lysine 79 (H3K79me1/2) is important for telomeric silencing and heterochromatin maintenance, but also promotes transcriptional elongation. It also correlates with replication initiation regions, and depletion of the sole H3K79 methyltransferase DOT1L can induce over-replication and genome instability (blue panel). Lysine 20 dimethylation of H4 (H4K20me2) is a common mark of euchromatin, and is bound by the Tudor domain of the DNA repair regulator 53BP1. 53BP1 binding promotes NHEJ by limiting DNA resection, suppressing RPA association and the recruitment of HR proteins (orange panel). H3 lysine 36 trimethylation (H3K36me3) increases along a gene body, peaking in the 3end of a transcribed region. Recent work has uncovered a role for H3K36me3 in regulating DNA mismatch repair (MMR), recruiting MutSa (Msh2-Msh6) through direct interaction with the Msh6 subunit even in the absence of DNA mismatches ([109], green panel). In the diagram of a gene body in the lower panel, black boxes mark exons while the thin black line represents non-coding DNA

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