RESUMO
The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Baculoviridae , Sequência de Bases , DNA/metabolismo , Proteína Quinase Ativada por DNA , Rearranjo Gênico , Camundongos , Dados de Sequência Molecular , Recombinação Genética , SpodopteraRESUMO
The ability of DNA oligonucleotides, neutral peptide nucleic acids (PNAS), and oligonucleotide conjugates to hybridize to inverted repeat sequences within supercoiled double-stranded DNA by Watson-Crick base-pairing is examined. PNAs and oligonucleotide conjugates initiate and maintain strand invasion under more stringent conditions than do unmodified DNA oligonucleotides. PNAs hybridize rapidly and, once bound, hold open a target site allowing oligonucleotides to base-pair to the displaced strand under conditions that would otherwise preclude hybridization. The ability to manipulate hybridization efficiency through different options for the alteration of oligomer charge should have important implications for optimizing sequence-specific recognition of DNA.
Assuntos
DNA de Cadeia Simples/química , DNA/química , Oligonucleotídeos/química , Oligopeptídeos/genética , Sequência de Aminoácidos , Composição de Bases , DNA/genética , DNA de Cadeia Simples/genética , Eletroforese em Gel de Ágar , Dados de Sequência Molecular , Mutação/genética , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Oligopeptídeos/química , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
V(D)J recombination is targeted by recombination signal sequences (RSS) located immediately adjacent to immune receptor gene segments. While the RSS flanking D(H) segments appear to be equivalent, they are not randomly utilized. During D(H) to J(H) rearrangement, the 3' D(H) RSS is virtually exclusively utilized, suggesting that the 3' D(H) RSS could simply be a better target for the recombinase. However, when we examined V(H) to D(H) (without J(H)) rearrangements, we found that the preference for D(H) RSS use changes, so that the 5' D(H) RSS are preferred. This suggests that the 3' D(H) RSS are not simply superior targets for the V(D)J recombinase, but instead that certain 12/23-bp spacer RSS combinations work better together to target recombination than do others. We have analyzed a series of artificial recombination substrates to delineate cis sequences that affect D(H) RSS selection. Our data suggest that coding sequences adjacent to the D(H) RSS, flanking sequences outside the D(H) gene segment itself, and recombination partner all affect D(H) RSS targeting.
