RESUMO
Three mammalian genes encoding DNA ligases--LIG1, LIG3, and LIG4--have been identified. Genetic, biochemical, and cell biology studies indicate that the products of each of these genes play a unique role in mammalian DNA metabolism. Interestingly, cell lines deficient in either DNA ligase I (46BR.1G1) or DNA ligase III (EM9) are sensitive to simple alkylating agents. One interpretation of these observations is that DNA ligases I and III participate in functionally distinct base excision repair (BER) subpathways. In support of this idea, extracts from both DNA ligase-deficient cell lines are defective in catalyzing BER in vitro and both DNA ligases interact with other BER proteins. DNA ligase I interacts directly with proliferating cell nuclear antigen (PCNA) and DNA polymerase beta (Pol beta), linking this enzyme with both short-patch and long-patch BER. In somatic cells, DNA ligase III alpha forms a stable complex with the DNA repair protein Xrcc1. Although Xrcc1 has no catalytic activity, it also interacts with Pol beta and poly(ADP-ribose) polymerase (PARP), linking DNA ligase III alpha with BER and single-strand break repair, respectively. Biochemical studies suggest that the majority of short-patch base excision repair events are completed by the DNA ligase III alpha/Xrcc1 complex. Although there is compelling evidence for the participation of PARP in the repair of DNA single-strand breaks, the role of PARP in BER has not been established.
Assuntos
DNA Ligases/fisiologia , Reparo do DNA/fisiologia , Isoenzimas/fisiologia , Animais , Células CHO , Linhagem Celular , Clonagem Molecular , Cricetinae , Cricetulus , Dano ao DNA , DNA Ligases/deficiência , DNA Ligases/genética , Reparo do DNA/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Genes , Teste de Complementação Genética , Humanos , Isoenzimas/deficiência , Isoenzimas/genética , Substâncias Macromoleculares , Mamíferos/genética , Mamíferos/metabolismo , Fenótipo , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif [1]. Here we examine the biological significance of this interaction. Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity or its interaction with DNA polymerase beta. In contrast, the loss of PCNA binding severely compromised the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site was also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells were defective in long-patch, but not short-patch, base-excision repair (BER). Our results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism.
Assuntos
DNA Ligases/metabolismo , Reparo do DNA , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , DNA Ligase Dependente de ATP , DNA Ligases/química , DNA Ligases/genética , Humanos , Mutagênese Sítio-Dirigida , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Ligação ProteicaRESUMO
In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.
