DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Abstract

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...

PrimPol‐mediated repriming facilitates replication traverse of DNA interstrand crosslinks

“…The genetic ablation of PRIMPOL caused a striking reduction in the frequency of traverse reactions but did not prevent them completely. Recently, an alternative pathway for ICL traverse involving the microcephaly associated DONSON protein instead of FANCM has been described (Zhang et al , 2020). We have shown here that PrimPol is epistatic within FANCM, so the DONSON pathway could be responsible in part for the remaining ICL traverse reactions in PRIMPOL KO cells.…”

Spotlight on the Replisome: Aetiology of DNA Replication-Associated Genetic Diseases

“…More recently, work by Zhang et al has provided evidence that DONSON is particularly enriched at replisomes in early replicating domains, suggesting a specific function in challenged and unchallenged conditions in euchromatin replication [99]. In summary, the aforementioned group of genetic syndromes caused by mutations in ATR-ATRIP, DNA2, CTIP, and the newly identified TRAIP and DONSON replisome components share a common mechanistic basis linked to defective maintenance of replication fork stability and/or ATR signalling (Figure 4).…”

Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin

“…These results were consistent with DONSON being retained on replisomes transiently proximal to ICLs, unlike the GINS proteins. Furthermore, following ATR inhibition there was an accumulation of DONSON containing replisomes stalled at ICLs (Zhang et al, 2020).…”

“…Thus, there were separate and distinguishable stressed replisomes containing either DONSON or FANCM but not both. Furthermore, DONSON clearly had a different role than FANCM because it was associated with both stressed and unstressed replisomes while FANCM was associated only with stressed replisomes (Zhang et al, 2020). This argued against the assumption of a single species of stressed replisome and raised the question: Do these different replisome complexes exist in the same cell at the same time?…”