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Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

USP1-associated factor 1 (UAF1) is an integral component of the RAD51-associated protein 1 (RAD51AP1)-UAF1-ubiquitin-specific peptidase 1 (USP1) trimeric deubiquitinase complex. This complex acts on DNA-bound, monoubiquitinated Fanconi anemia complementation group D2 (FANCD2) protein in the Fanconi anemia pathway of the DNA damage response. Moreover, RAD51AP1 and UAF1 cooperate to enhance homologous DNA pairing mediated by the recombinase RAD51 in DNA repair via the homologous recombination (HR) pathway. However, whereas the DNA-binding activity of RAD51AP1 has been shown to be important for RAD51-mediated homologous DNA pairing and HR-mediated DNA repair, the role of DNA binding by UAF1 in these processes is unclear. We have isolated mutant UAF1 variants that are impaired in DNA binding and tested them together with RAD51AP1 in RAD51-mediated HR. This biochemical analysis revealed that the DNA-binding activity of UAF1 is indispensable for enhanced RAD51 recombinase activity within the context of the UAF1-RAD51AP1 complex. In cells, DNA-binding deficiency of UAF1 increased DNA damage sensitivity and impaired HR efficiency, suggesting that UAF1 and RAD51AP1 have coordinated roles in DNA binding during HR and DNA damage repair. Our findings show that even though UAF1's DNA-binding activity is redundant with that of RAD51AP1 in FANCD2 deubiquitination, it is required for efficient HR-mediated chromosome damage repair., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Liang et al.)

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes., (© 2018 Wang et al.)

Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G2/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN(610) with either DDD(610) or DDE(610) significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN(610) → DDD(610), which restores the ancestral catalytic site, results in loss of function in Metnase.

A novel member of the winged helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis, was isolated and characterized. Putative translation of the complete cDNA revealed the winged helix DNA binding domain to be centrally located within the protein, with regions on either side that contain known transcriptional regulatory motifs. Extensive Northern analysis of Genesis found that the message was exclusively expressed in embryonic stem cells or their malignant equivalent, embryonal carcinoma cells. The Genesis transcript was down-regulated when these cells were stimulated to differentiate. DNA sequences that Genesis protein would interact with were characterized and were found to contain a consensus similar to that found in an embryonic stem cell enhancer sequence. Co-transfection experiments revealed that Genesis is a transcriptional repressor. Genesis mapped to mouse chromosome 4 in a region syntenic with human chromosome 1p31, a site of nonrandom abnormalities in germ cell neoplasia, neuroblastoma, and acute lymphoblastic leukemia. Genesis is a candidate for regulating the phenotype of normal or malignant embryonic stem cells.