SUMMARY Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

25Duplication of mammalian genomes requires replisomes to overcome numerous impediments 26 during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of 27 replication stress characterize mixed populations of challenged and unchallenged replication forks, 28 averaged across S phase, and model a single species of "stressed" replisome. However, in cells 29 containing potent obstacles to replication, we find two different lesion proximal replisomes. One 30 is bound by the DONSON protein and is more frequent in early S phase, in regions marked by 31 euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S 32 phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. CHIP-33 seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions 34 that replicate early and the skew of the latter towards regions that replicate late. 35Introduction 36 Eukaryotic replisomes are multiprotein complexes consisting, minimally, of the CMG 37 helicase [MCM2-7 (M), CDC45 (C), and GINS (go, ichi, ni, san) proteins (G)] which forms a ring 38 around the leading strand template. Other components include the pol α, ε, and δ polymerases, 39 MCM10, and a few accessory factors [1][2][3][4][5][6][7] . The identification and characterization of the minimal 40 components of biochemically active replisomes, the result of decades of extraordinary work from 41 multiple laboratories, necessarily reflects studies with deproteinized model DNA substrates under 42 carefully controlled conditions. However, in vivo there are hundreds of replisome associated 43 proteins 8-12 . Presumably this reflects the multiple layers of complexity that characterize replication 44 of the genome in living cells. For example, three dimensional analyses of chromosome structure 45 demonstrate two major domains. The A compartment contains euchromatin, which is accessible, 46 transcriptionally active, and marked by specific histone modifications, such as H3K4me3. The B 47 compartment, which is more condensed, contains inactive genes, many repeated elements, and is 48 associated with different histone modifications, including H3K9me3 13 . In addition to the structural 49 distinctions, regions of the genome are also subject to temporal control of replication during S 50 phase. Sequences in Compartment A tend to replicate early in S phase, while those in B are 51 duplicated in late S phase 14,15 . 52 Other influential effectors of replisome composition are the frequent encounters with 53 impediments, that stall or block either the progress of the CMG helicase or DNA synthesis. These 54 3 include alternate DNA structures, protein: DNA adducts, DNA covalent modifications introduced 55 by endogenous or endogenous reactants, depleted nucleotide precursor pools, etc. Replication 56 stress activates the ATR (ATM-and Rad3-related) kinase, with hundreds of substrates, including 57 MCM proteins [16][17][18] , and stimulates the recruitment of numerous facto...

Various stem cells in the body are tasked with maintaining tissue homeostasis throughout the life of an organism and thus must be resilient to intrinsic and extrinsic challenges such as infection and injury. Crucial to these challenges is genome maintenance because a high mutational load and persistent DNA lesions impact the production of essential gene products at proper levels and compromise optimal stem cell renewal and differentiation. Genome maintenance requires a robust and well-regulated DNA damage response suited to maintaining specific niches and tissues. In this review, we explore the similarities and differences between diverse stem cell types derived from (or preceding) all germ layers, including extraembryonic tissues. These cells utilize different strategies, including implementation of robust repair mechanisms, modulation of cell cycle checkpoints best suited to eliminating compromised cells, minimization of cell divisions, and differentiation in response to excessive damage. Expected final online publication date for the Annual Review of Genetics, Volume 56 is November 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling–double strand breaks, reversed forks, or genomic rearrangements–there is limited understanding of the determinants of replisome stalling vs. continued progression. Although many proteins are recruited to stalled replisomes, current models assume a single species of “stressed” replisome, independent of genomic location. Here we describe our approach to visualizing replication fork encounters with the potent block imposed by a DNA interstrand crosslink (ICL) and our discovery of an unexpected pathway of replication restart (traverse) past an intact ICL. Additionally, we found two biochemically distinct replisomes distinguished by activity in different stages of S phase and chromatin environment. Each contains different proteins that contribute to ICL traverse.