The reaction mechanism of Xenopus (6-4) photolyase was investigated using several mutant enzymes. In the active site, which is homologous between the cis,syn-cyclobutane pyrimidine dimer and (6-4) photolyases, four amino acid residues that are specific to (6-4) photolyase, Gln(288), His(354), Leu(355), and His(358), and two conserved tryptophans, Trp(291) and Trp(398), were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp(291) and Trp(398) mutants retained some activity. Taking the pH profile of the (6-4) photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.

The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32 Plabeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54°and 57°in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle.

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3 termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative "3 terminus binding pocket" present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses.Progression of replication forks is stalled by a variety of internal and external causes, including DNA damage and depletion of precursors for DNA synthesis. The arrested replication forks elicit checkpoint responses that enable cells to repair damage, restore replication forks, and restart DNA replication (1, 2). A number of eukaryotic gene products have been identified that participate in this process. The initial phase of this response is the recognition of the arrested forks by a sensor protein, which may be recruited to the site of the fork arrest and transduce various signals for further downstream cellular responses. However, the nature of the protein that detects the stalled replication forks and the mechanism by which they are recognized are still unclear.In Escherichia coli, PriA, a DEXH-type DNA helicase originally discovered as a protein essential for replication of a small single-stranded phage DNA, X174, is believed to play a key role in rescuing the stalled replication forks (1, 3-5). The priA null cells display numerous phenotypes, including low viability and sensitivity to DNA damaging agents such as UV, ␥-ray, and mitomycin C (6 -10). These defects may reflect a critical role played by PriA in resumption of DNA replication after arrest of ongoing replication forks (11). In E. coli, in fact, arrest of ongoing replication forks induces a recombination-dependent mode of DNA replication, known as inducible or constitutive stable DNA replication (iSDR 1 or cSDR, respectively), which requires PriA protein (8). It was shown that the X174-type primosome could be assembled on a model D-loop structure in vitro (12, 13). It has been proposed that PriA may recognize recombination intermediates generated as a result of fork arrest as well as the arrested replication fork per se and facilitate reassembly of replication forks (8,14,15). Consistent with this, PriA recognizes and binds specifically to DNA structures such as those mimicking D-loop (intermediates of homologous recombination reactions) or arrested replication forks (16 -18). However, it has not been known what structural features of D-loop or arrested DNA replication fork structures are recognized by PriA. In this report, w...

No abstract

The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated β2-adrenoceptor (β2AR) and the phosphorylated β2AR–β-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M2155.54 and M2796.41, located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the β-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables β-arrestin to recognize dozens of GPCRs.