Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

Abstract

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA

“…Upon expression of human TREX1, the insect cells were resuspended in a hypotonic buffer (20 mM Hepes-KOH, pH 7.5, 0.5 mM MgCl 2 , 5 mM KCl, 5 mM ␤-mercaptoethanol, and a mixture of protease inhibitors (19)). Nuclei were separated from the cytoplasm of insect cells by homogenization on ice using 10 strokes of a Dounce homogenizer followed by centrifugation at 4,200 rpm (Beckman Coulter rotor JLA-10.500) at 4°C for 6 min.…”

FANCA and contribution of studies from Asian populations to the understanding of fanca mediated Fanconi anemia

“…FANCA protein has the ability to recognizing ICLs and triggers the subsequent cascade of proteins to resolve the damage. In vitro study of Benitez and his colleagues have shown that on the stalled replication fork or ICL sites, FANCA recruits the heterodimer MUS81-EME1 complex that subsequently incises at the 3' side of the leading strand (BENITEZ et al, 2013). Interestingly, FANCA protein acts both as activator/enhancer or inhibitor of MUS81-EME1 because various studies elucidate that upon encountering the ICL damages, MUS81-EME1 activated and recruited by FANCA for potent and delicate ICL incision (BENITEZ et al, 2013).…”

“…In vitro study of Benitez and his colleagues have shown that on the stalled replication fork or ICL sites, FANCA recruits the heterodimer MUS81-EME1 complex that subsequently incises at the 3' side of the leading strand (BENITEZ et al, 2013). Interestingly, FANCA protein acts both as activator/enhancer or inhibitor of MUS81-EME1 because various studies elucidate that upon encountering the ICL damages, MUS81-EME1 activated and recruited by FANCA for potent and delicate ICL incision (BENITEZ et al, 2013). Furthermore, it has been demonstrated that MUS81-EME1 also resolves Holliday junction, the final step of FA pathway in damage repairing (HOLLINGSWORTH et al 2004;KIM et al, 2013).…”

“…There is a growing myriad of FANCA interacting proteins each having a role in different mechanism. For example interaction of FANCA with alpha spectrin II (αIISP) is required to act as scaffold protein in FA repair activities (MCMAHON et al, 1999), with MUS81-EME1 complex for repair of DNA ICLs (BENITEZ et al, 2013), with FEN1 for stabilizing the replication machinery while working on lagging strand (QIAN et al, 2013a;QIAN et al, 2013b;PALOVCAK et al, 2017a), with HES1 for stabilizing the core complex members (TREMBLAY et al, 2008), IκB Kinase-2 (IKK2) interaction for cellular stress response signaling pathway (OTSUKI et al, 2002b), and with sorting nexin 5 (SNX5) for translocation between organelles (OTSUKI et al, 1999). FANCA may engages with BRG1(Brahma-related gene 1) to alleviate the effects of ROS by enhancing the binding ability of BRG1 with antioxidant gene promotors (OTSUKI et al, 2001), with NEK2 to preserve centromere integrity, and with CENP-E for chromosomal alignment at the spindle equator and congregation during mitosis (DU et al 2009).…”

Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli

“…XPF-ERCC1, a nuclease subunit of the nucleotide excision repair complex, is postulated to function in a replicationcoupled pathway of DNA interstrand cross-link repair that is separate from its role in the general nucleotide excision repair pathway (34,35). Other nucleases, such as MUS81-EME1, SLX1 to SLX4, and FAN1, are also proposed to participate in DNA interstrand cross-link repair based upon studies using oligonucleotide or plasmid substrates in cell extracts (36)(37)(38)(39)(40)(41)(42). The molecular mechanisms and intermediates for both replication-coupled and global genomic repair pathways in eukaryotes remain speculative.…”