The replisome, the molecular machine dedicated to copying DNA, encounters a variety of obstacles during S phase. Without a proper response to this replication stress, the genome becomes unstable, ...leading to disease, including cancer. The immediate response is localized to the stalled replisome and includes protection of the nascent DNA. A number of recent studies have provided insight into the factors recruited to and responsible for protecting stalled replication forks. In response to replication stress, the SNF2 family of DNA translocases has emerged as being responsible for remodeling replication forks in vivo. The protection of stalled replication forks requires the cooperation of RAD51, BRCA1, BRCA2, and many other DNA damage response proteins. In the absence of these fork protection factors, fork remodeling renders them vulnerable to degradation by nucleases and helicases, ultimately compromising genome integrity. In this review, we focus on the recent progress in understanding the protection, processing, and remodeling of stalled replication forks in mammalian cells.
Telomeres are essential for genome stability in all eukaryotes. Changes in
telomere functions and the associated chromosomal abnormalities have been
implicated in human aging and cancer. Telomeres ...are composed of repetitive
sequences that can be maintained by telomerase, a complex containing a reverse
transcriptase (hTERT in humans and Est2 in budding yeast), a template RNA
(hTERC in humans and Tlc1 in yeast), and accessory factors (the Est1 proteins
and dyskerin in humans and Est1, Est3, and Sm proteins in budding yeast).
Telomerase is regulated in
cis
by proteins that bind to telomeric DNA.
This regulation can take place at the telomere terminus, involving
single-stranded DNA-binding proteins (POT1 in humans and Cdc13 in budding
yeast), which have been proposed to contribute to the recruitment of telomerase
and may also regulate the extent or frequency of elongation. In addition,
proteins that bind along the length of the telomere (TRF1/TIN2/tankyrase in
humans and Rap1/Rif1/Rif2 in budding yeast) are part of a negative feedback
loop that regulates telomere length. Here we discuss the details of telomerase
and its regulation by the telomere.
Celotno besedilo
Dostopno za:
CMK, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most ...knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
HMCES, a recently discovered but ancient protein, covalently attaches to damaged single-stranded DNA and shields it from nucleases. Rua-Fernandez and colleagues now show that HMCES catalyzes its own ...recycling, permitting normal growth and non-mutagenic DNA repair.1
HMCES, a recently discovered but ancient protein, covalently attaches to damaged single-stranded DNA and shields it from nucleases. Rua-Fernandez and colleagues now show that HMCES catalyzes its own recycling, permitting normal growth and non-mutagenic DNA repair.
Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid ...tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
LINE-1 retrotransposon overexpression is a hallmark of human cancers. We identified a colorectal cancer wherein a fast-growing tumor subclone downregulated LINE-1, prompting us to examine how LINE-1 ...expression affects cell growth. We find that nontransformed cells undergo a TP53-dependent growth arrest and activate interferon signaling in response to LINE-1. TP53 inhibition allows LINE-1
cells to grow, and genome-wide-knockout screens show that these cells require replication-coupled DNA-repair pathways, replication-stress signaling and replication-fork restart factors. Our findings demonstrate that LINE-1 expression creates specific molecular vulnerabilities and reveal a retrotransposition-replication conflict that may be an important determinant of cancer growth.
► The Fanconi anemia (FA) is a bone marrow and cancer predisposition syndrome. ► The FA pathway is responsible for repair of interstrand DNA crosslinks. ► FA core complex ubiquitylates FANCI and ...FANCD2 which localize to chromatin and direct repair. ► Ubiquitin interaction motifs are used to recruit nucleases to sites of damage. ► Deubiquitylation plays an important role in terminating DNA damage signaling.
The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability.
DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) ...pathways. Pathogenic variants in the essential gene
when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of
DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying
DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.
SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in ...Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.
•Mutational analysis of the Fanconi anemia nuclease scaffold SLX4/FANCP reveals lesion-dependent functional requirements for XPF, MUS81, and SLX1 in DNA repair.•The UBZ domain and SLX4-XPF complex are critical for interstrand cross-link repair and the SLX4-MUS81 complex repairs CPT and PARP inhibitor-induced damage.
Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that ...generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.
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