John B. Leppard

Physical and Functional Interaction between DNA Ligase IIIα and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

ABSTRACT The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIα, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIα but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIα-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.

Human DNA topoisomerase I: relaxation, roles, and damage control.

Human DNA topoisomerase I is an essential enzyme involved in resolving the torsional stress associated with DNA replication, transcription, and chromatin condensation. The catalytic cycle of the enzyme consists of DNA cleavage to form a covalent enzyme–DNA intermediate, DNA relaxation, and finally, religation of the phosphate backbone to restore the continuity of the DNA. Structure/function studies have elucidated a flexible enzyme that relaxes DNA through coordinated, controlled movements of distinct enzyme domains. The cellular roles of topoisomerase I are apparent throughout the nucleus, but the concentration of processes acting on ribosomal DNA results in topoisomerase I accumulation in the nucleolus. Although the activity of topoisomerase I is required in these processes, the enzyme can also have a deleterious effect on cells. In the event that the final religation step of the reaction cycle is prevented, the covalent topoisomerase I–DNA intermediate becomes a toxic DNA lesion that must be repaired. The complexities of the relaxation reaction, the cellular roles, and the pathways that must exist to repair topoisomerase I-mediated DNA damage highlight the importance of continued study of this essential enzyme.

SCAN1 mutant Tdp1 accumulates the enzyme–DNA intermediate and causes camptothecin hypersensitivity

Tyrosyl‐DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl‐3′ phosphate linkage found in topoisomerase I–DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss‐of‐function mutation, we show here that this mutation reduces enzyme activity ∼25‐fold and importantly causes the accumulation of the Tdp1–DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I–DNA complexes by Tdp1 unexpectedly generates a new protein–DNA complex with an apparent half‐life of ∼13 min that, in addition to the unrepaired topoisomerase I–DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I‐specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT‐induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.

Completion of base excision repair by mammalian DNA ligases

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.

DNA ligase III is recruited to DNA strand breaks by a zinc finger motif homologous to that of poly (ADP-ribose) polymerase. Identification of two functionally distinct DNA binding regions within DNA ligase III

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activityin vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivothe DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.

Antibody discovery ex vivo accelerated by the LacO/LacI regulatory network.

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.