SUMMARY FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here we show that FANCM forms a conserved DNA remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA remodeling complex that protects replication forks from yeast to human.

Fanconi anemia (FA) is a genetically heterogeneous chromosome instability syndrome associated with congenital abnormalities, bone marrow failure, and cancer predisposition. Eight FA proteins form a nuclear core complex, which promotes tolerance of DNA lesions in S phase, but the underlying mechanisms are still elusive. We reported recently that the FA core complex protein FANCM can translocate Holliday junctions. Here we show that FANCM promotes reversal of model replication forks via concerted displacement and annealing of the nascent and parental DNA strands. Fork reversal by FANCM also occurs when the lagging strand template is partially single-stranded and bound by RPA. The combined fork reversal and branch migration activities of FANCM lead to extensive regression of model replication forks. These observations provide evidence that FANCM can remodel replication fork structures and suggest a mechanism by which FANCM could promote DNA damage tolerance in S phase.fanconi anemia ͉ replication fork A variety of structural and chemical alterations in DNA can hinder the progression of replication forks and precipitate the formation of gross chromosomal rearrangements. These hurdles impose distinct structural constraints in the template DNA, which elicit the action of diverse lesion bypass or lesion tolerance pathways (1, 2). Covalent links between complementary DNA strands constitute a unique challenge to replicating cells, because they preclude strand separation and, hence, completely block fork progression. In mammalian cells, the repair of DNA interstrand cross-links (ICLs) is thought to take place during S phase (3). The exact mechanism of repair is unknown, but it seems to involve the interplay of different pathways, with the homologous recombination machinery, translesion DNA polymerases, and the Fanconi anemia (FA) pathway all being required for ICL tolerance (4).FA is a genetically heterogeneous inherited disorder, which combines congenital abnormalities, bone marrow failure, and a marked cancer predisposition (5-8). FA cells are prone to spontaneous and damage-induced chromosomal aberrations and are notoriously hypersensitive to DNA interstrand cross-linking agents. FA proteins can be classified into three groups (8). Group I includes FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM. These eight FA proteins form a nuclear core complex (9-11) whose integrity is required for the conjugation of a ubiquitin moiety to the group II proteins, FANCI and FANCD2 (12,13). Group III consists of FANCD1 (BRCA2), FANCN (PALB2), and FANCJ (BRIP1), which do not play a role in FANCD2 monoubiquitination. BRCA2 regulates formation of RAD51 nucleoprotein filaments during homologous recombination (14, 15), PALB2 is necessary for the correct association of BRCA2 with chromatin (16), and BRIP1 is a BRCA1-associated DNA helicase that contributes to homologous recombination and cross-link repair (17, 18).The FA core complex protein FANCM can specifically bind to model replication forks and Holliday junctions and move the...

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Divalent metal transporter1 (DMT1; also known as DCT1 or NRAMP2) is an important component of the cellular machinery responsible for dietary iron absorption in the duodenum. DMT1 is also highly expressed in the kidney where it has been suggested to play a role in urinary iron handling. In this study, we determined the effect on renal DMT1 expression of feeding an iron-restricted diet (50 mg/kg) or an iron-enriched diet (5 g/kg) for 4 wk and measured urinary and fecal iron excretion rates. Feeding the low-iron diet caused a reduction in serum iron concentration and fecal iron output rate with an increase in renal DMT1 expression. Feeding an ironenriched diet had the converse effect. Therefore, DMT1 expression in the kidney is sensitive to dietary iron intake, and the level of expression is inversely related to the dietary iron content. Changes in DMT1 expression occurred intracellularly in the proximal tubule and in the apical membrane and subapical region of the distal convoluted tubule. Increased DMT1 expression was accompanied by a decrease in urinary iron excretion rate and vice versa when DMT1 expression was reduced. Together, these findings suggest that modulation of renal DMT1 expression may influence renal iron excretion rate. serum iron level; kidney; iron regulatory protein; SLC11A2; NRAMP2 IRON IS AN ESSENTIAL metal for life because it is a key constituent of a family of fundamental proteins, which includes hemoglobin, cytochromes, and NADH-coenzyme Q reductase. Maintaining the correct balance of iron is paramount to health because iron deficiency or excess results in morbidity and mortality. The molecular characterization of membrane-bound iron transporter proteins, in particular divalent metal transporter1 (DMT1; 9), also known as DCT1 (14) or NRAMP2 (13), has shed new light on some of the mechanisms of body iron homeostasis. DMT1 is the product of the

Our results indicate that the G185R mutation of DMT1 causes protein instability in the kidneys of b/b rats. Given that +/b and b/b rats excrete comparable amounts of iron, the lack of DMT1 protein is compensated by an alternative, yet to be identified, mechanism.