Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3-5 exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3 singlestrand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time-and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner. INTRODUCTIONThe MRN complex, consisting of Mre11, Rad50 and NBS1, has diverse functions in DNA damage recognition (Petrini and Stracker, 2003), DNA replication (Costanzo et al., 2001), cell cycle checkpoint activation (Grenon et al., 2001), nonhomolgous end joining (Paull and Gellert, 2000), and telomere maintenance (Wu et al., 2007). The Mre11 complex binds DNA double-strand breaks (DSBs) soon after they are formed implicating it in DNA damage detection . Furthermore, the complex can tether linear duplex molecules (de Jager et al., 2001), and it is able to bridge broken DNA ends or sister chromatids (van den Bosch et al., 2003). Mre11 has 3Ј-5Ј exonuclease activity and endonuclease activity (Paull and Gellert, 1999), suggesting a role in the processing of DNA ends into forms that can be recognized by cell cycle checkpoint and DNA repair proteins (Paull and Gellert, 1999;Lee and Paull, 2005;Jazayeri et al., 2006). However, precise cellular roles of the Mre11 complex have been difficult to establish, because null mutations of all components of the complex are lethal to vertebrate cells (Luo et al., 1999;Yamaguchi-Iwai et al., 1999;Zhu et al., 2001).There are several lines of evidence implicating the MRN complex in DNA replication. The complex associates with chromatin and colocalizes with proliferating cell nuclear antigen (PCNA) throughout S phase (Maser et al., 2001). In addition, chromatin loading of Mre11 is enhanced by fork stalling, suggesting that the complex is loaded at the replication fork (Mirzoeva and Petrini, 2003). Depletion of Mre11 from DT40 or Xenopus leads to increased chromosomal breaks and accumulation of DSBs during DNA replication (Yamaguchi-Iwai et al., 1999;Costanzo et...

As a newly-identified protein post-translational modification, malonylation is involved in a variety of biological functions. Recognizing malonylation sites in substrates represents an initial but crucial step in elucidating the molecular mechanisms underlying protein malonylation. In this study, we constructed a deep learning (DL) network classifier based on long short-term memory (LSTM) with word embedding (LSTMWE) for the prediction of mammalian malonylation sites. LSTMWE performs better than traditional classifiers developed with common pre-defined feature encodings or a DL classifier based on LSTM with a one-hot vector. The performance of LSTMWE is sensitive to the size of the training set, but this limitation can be overcome by integration with a traditional machine learning (ML) classifier. Accordingly, an integrated approach called LEMP was developed, which includes LSTMWE and the random forest classifier with a novel encoding of enhanced amino acid content. LEMP performs not only better than the individual classifiers but also superior to the currently-available malonylation predictors. Additionally, it demonstrates a promising performance with a low false positive rate, which is highly useful in the prediction application. Overall, LEMP is a useful tool for easily identifying malonylation sites with high confidence. LEMP is available at http://www.bioinfogo.org/lemp.

RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.Helicases are ubiquitous enzymes essential in all stages of DNA and RNA metabolism, including replication, transcription, recombination, and repair (1, 2). They are motor proteins that generally translocate along one or both strands of dsDNA, 1 dsRNA, or a DNA-RNA hybrid in an ATP-dependent manner and separate some or all parts of the molecule into its component strands. Some enzymes are highly processive, driving strand separation for long distances without dissociating from the template, a property that is especially important for enzymes involved in chromosome replication (3).A variety of mechanisms has evolved to ensure processivity. The prokaryotic helicase DnaB, for example, forms a hexameric ring that completely encircles the DNA, which allows onedimensional motion while preventing dissociation (4, 5). Such a strategy is common to many of the hexameric helicases (6). Other helicases are thought to achieve the same effect by interacting with the "sliding clamp" that encircles the DNA strands during replication. Examples include Wrn and Rrm3, which have been shown to have enhanced processivity in the presence of PCNA (7,8). The RecC component of the RecBCD helicase has no helicase activity in itself, but its role may be similar to a sliding clamp, preventing dissociation of the RecBCD complex from the DNA (9 -11).A second strategy is for the helicase to have two binding sites, such that one site can release and move along the strand while the second stays bound. This can be achieved by having a dimeric structure, with one binding site per monomer and has led to the proposal of the "hand over hand" or "rolling" method. Rep helicase is thought to translocate using this mechanism (12). The Rep dimer binds to DNA, with one monomer behind the other. ATP hydrolysis alters the conformation of the helicase, radically changing the position of one monomer relative to the other, as well as causing a change in its nucleic acid binding properties (12)(13)(14). This movement means the second monomer can now bind the DNA in front of the first monomer, and thus translocation i...