Time for remodeling: SNF2-family DNA translocases in replication fork metabolism and human disease

Abstract

Over the course of DNA replication, DNA lesions, transcriptional intermediates and protein-DNA complexes can impair the progression of replication forks, thus resulting in replication stress. Failure to maintain replication fork integrity in response to replication stress leads to genomic instability and predisposes to the development of cancer and other genetic disorders. Multiple DNA damage and repair pathways have evolved to allow completion of DNA replication following replication stress, thus preserving genomic integrity. One of the processes commonly induced in response to replication stress is fork reversal, which consists in the remodeling of stalled replication forks into four-way DNA junctions. In normal conditions, fork reversal slows down replication fork progression to ensure accurate repair of DNA lesions and facilitates replication fork restart once the DNA lesions have been removed. However, in certain pathological situations, such as the deficiency of DNA repair factors that protect regressed forks from nuclease-mediated degradation, fork reversal can cause genomic instability. In this review, we describe the complex molecular mechanisms regulating fork reversal, with a focus on the role of the SNF2-family fork remodelers SMARCAL1, ZRANB3 and HLTF, and highlight the implications of fork reversal for tumorigenesis and cancer therapy.

Keywords: Cancer; DNA damage; Genomic instability; Innate Immunity; Replication fork remodeling; Replication stress.

Figure 1.. Simplified schematics of the mechanisms of replication fork restart upon replication stress

When the replication machinery encounters a replication block (stop sign), replication fork progression stalls, leading to the formation of single-stranded DNA (ssDNA) that is rapidly coated by the RPA trimer (I). Restart of stalled forks can occur through lesion bypass by translesion synthesis or fork repriming (II-III). Translesion synthesis allows the direct bypass of the DNA lesion (II), while fork repriming promotes the restart of DNA synthesis downstream of the lesion (III). Alternative to lesion bypass, stalled forks can undergo reversal mediated by fork remodelers, including the SNF2-family members SMARCAL1, ZRANB3 and HLTF (IV). Fork reversal slows down replication fork progression, thus allowing sufficient time for repair of the lesion. Reset of reversed forks by the RECQ1 helicase can then promote the restart of DNA synthesis once the lesion has been removed (V). Fork reversal could also directly promote the bypass of the DNA lesion by enabling DNA synthesis on the nascent DNA strand of the sister chromatid (green) through template switching (not shown). Following fork reversal, the exposed ends of the regressed fork are stabilized by RAD51 to limit fork resection by the nucleases MRE11, DNA2 and EXO1 and protect the integrity of the fork (IV). Limited fork resection can facilitate fork restart through the reset of reversed forks or HR-dependent strand invasion catalyzed by the 3’-end of the resected regressed arm (not shown). BRCA1/2, RAD51 paralogs and Fanconi anemia proteins cooperate with RAD51 to control fork resection and maintain fork protection. Defective fork protection causes extensive fork resection and processing of the regressed fork by the endonucleases SLX4 and MUS81, leading to fork collapse (VI). Fork collapse can also result from MUS81-mediated cleavage of persistently arrested replication forks or from forks encountering single-strand DNA breaks. Collapsed forks are repaired by HR-mediated fork restart pathways dependent on RAD51 and/or RAD52 (VI-VIII). Key factors involved in each pathway and described in the main text are indicated.

Figure 2.. The SNF2 family of proteins

Depiction of SNF2-family group relationships, based on alignments of the DNA translocase domain (adapted from Flaus et al. [98]). Schematic representations (not to scale) of protein domains characteristic of each subfamily in humans are shown. All SNF2-family members contain a DNA translocase domain consisting of DEXDc and HELICc domains. The SNF2-like subfamily consists of ISWI-like, CHD-like and SWI/SNF-like groups. The ISWI-like group includes SMARCA1 (1054 aa) and SMARCA5 (1052 aa). The CHD-like group includes CHD1 (1710 aa), CHD2 (1828 aa), CHD3 (2000 aa), CHD4 (1912 aa), CHD5 (1954 aa), CHD6 (2715 aa), CHD7 (2997 aa), CHD8 (2581 aa), CHD9 (2897 aa) and CHD1L (897 aa). CHD3, CHD4 and CHD5 have N-terminal plant homeodomain (PHD) fingers not represented in the schematic, while CHD1L lacks the N-terminal chromodomains and contains a C-terminal macrodomain (not shown). The SWI/SNF-like group includes SMARCA2 (1590 aa) and SMARCA4 (1647 aa). HELLS (838 aa) is closely related to the SNF2-like subfamily, but it does not contain domains typical of the members of this subfamily (not shown). The INO80-like subfamily includes INO80 (1556 aa), SRCAP (3230 aa) and EP400 (3159 aa), which contain additional A/T hook and SANT domains, respectively, and SMARCAD1 (1026 aa), which lacks the HSA domain and contains two N-terminal coupling of ubiquitin conjugation to ER degradation (CUE) motifs not represented in the schematic. The SSO1653-like subfamily includes BTAF1 (1849 aa), which contains multiple HEAT/ARM repeats, and ERCC6L (1250 aa), which harbors two tetratricopeptide repeat (TPR) motifs and a PICH family domain (PFD) of unclear function not represented in the schematic. The RAD54-like subfamily includes RAD54 (747 aa) and ATRX (2492 aa), which contains an N-terminal ATRX-DNMT1-DNMT1L (ADD) domain not shown. The RAD5/16-like subfamily includes SHPRH (1683 aa), which contains a linker histone H1/H5 (H15) domain in addition to the domains represented in the figure, and HLTF (1009 aa). Finally, the SMARCAL1-like subfamily includes SMARCAL1 (954 aa) and ZRANB3 (1079 aa). SMARCAL1, ZRANB3 and HLTF (in red) are the only SNF2-family members that have been currently shown to regress stalled replication forks in vivo. HLTF contains a RING domain that mediates the interaction between HLTF and substrates to ubiquitinate. SMARCAL1 harbors an RPA2-binding motif to directly interact with RPA. ZRANB3 contains a PCNA-interacting protein (PIP) motif, an AlkB homolog 2 PCNA interacting motif (APIM) and a NPL4 zinc-finger (NZF) motif that cooperate to bind polyubiquitinated PCNA. The HIRAN, HARP and SRD (substrate recognition) domains of HLTF, SMARCAL1, and ZRANB3, respectively, enable structure specific DNA binding. The HNH motif of ZRANB3 is a nuclease domain.

Figure 3.. Cellular effects induced by replication stress

Failure to complete DNA replication due to replication stress results in under-replication and sister-chromatid bridge formation. If the bridges are not resolved during anaphase, the sister chromatids remain attached to each other, resulting in chromosome mis-segregation, lagging chromosome formation, or chromosome breakage. Chromosomal breakage and mis-segregation results in the formation of chromosomal aberrations. Chromosomal fragments or lagging chromosomes that fail to be incorporated into the daughter cell nucleus form micronuclei. Errors during DNA synthesis and DNA repair processes can result in the inaccurate duplication of the genome and mutagenesis. Cytoplasmic DNA fragments and micronuclei originating upon replication stress may be recognized by the cGAS-STING pathway, leading to the induction of interferons, immune stimulated genes (ISGs), and immune checkpoint factors (e.g., PD-L1). PD-L1 expression inhibits immune cell surveillance, leading to immune evasion and immunosuppression.

Figure 4.. Methods for studying replication fork transactions

Overview of the current techniques used to (1) monitor replication fork dynamics and visualize replication fork structures, (2) identify proteins associated with and localized on nascent DNA, (3) analyze locus-specific replication fork progression, (4) monitor genomic replication at fork barriers, and (5) reconstitute in vitro replication fork transactions with and without the presence of barriers. DNA fiber and DNA combing techniques enable the analysis of the progression of individual replication forks, while electron microscopy allows high-resolution detection of DNA intermediates and replication fork structures, such as reversed forks and ssDNA gaps. Protein identification and localization on newly synthesized DNA can be obtained by iPOND, PLA-based approaches, and super-resolution microscopy methods, such as STORM. Techniques, such as SMARD, nanopore-based sequencing methods (D-NAscent, Fork-seq), and optical mapping enable the study of replication fork dynamics at genomic loci of interest. DNA lesions labeled with chemically modified adducts and/or antibodies or site-specific replication fork barriers, such as the E. coli Tus-Ter system, allow the analysis of events occurring when replication forks encounter obstacles. In vitro studies of replication fork transactions can be conducted using Xenopus egg extracts, which enable the study of replication fork progression, with and without fork barriers. Similar studies can be performed using purified replication factors that reconstitute DNA synthesis in vitro. Purified proteins can also be utilized to detect enzymatic activities on fork-like substrates or define DNA transactions at single-molecule level using DNA curtains or optical tweezers technologies combined with fluorescence microscopy.