Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I.

Abstract

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A lymphoma (3, 32). Fibroblasts from the patient, 46BR, are hypersensitive to killing by several DNA-damaging agents and also by 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase (27,28). Moreover, 46BR cells are anomalously sensitive to induction of sister chromatid exchanges and show delayed strand break rejoining after DNA damage (10). Retarded joining of Okazaki fragments during DNA replication has also been reported for 46BR cells (10,15,17). These observations suggest a role for DNA ligase I in both lagging-strand DNA synthesis and DNA repair processes.One of the mutations in 46BR fibroblasts results in an inactivating Glu-566--Lys replacement within the highly conserved active site of the enzyme. The mutation in the other allele, Arg-771-+Trp, also occurs in a region of the protein showing marked evolutionary conservation (3). The latter mutation is inherited from the mother and is present in two brothers of the patient. All three heterozygotes are clinically normal (32 Arg-771--*Trp mutation on the activity of DNA ligase I, as well as the altered function of the mutant enzyme in in vitro assays for DNA repair and replication. MATERIALS AND METHODSCell lines. The simian virus 40-transformed human cell line 46BR. lGl was derived from the DNA ligase I-defective primary fibroblast strain 46BR (3) following transfection with pSV3gpt (15, 20). The simian virus 40-transformed line MRC5V1 was established from normal human fibroblasts (11). The cells were propagated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. HeLa cells were grown in suspension culture under standard conditions.Immunopurification of DNA ligase I from cell extracts. Frozen cell pellets were thawed and incubated for 30 min on ice in lysis buffer containing several protease inhibitors (1 ml/107 cells): 50 mM Tris-HCl (pH 8.5), 125 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 100 mM Na3PO4, 170 ,ug of phenylmethylsulfonyl fluoride per ml, 27 ,ug of aprotinin per ml, and 0.5 ,ug each of leupeptin, pepstatin, chymostatin, and tosyllysine chloromethylketone (TLCK) per ml. The lysate was centrifuged at 5,000 x g for 15 min at 4°C, and the supernatant was recovered. Ten microliters of rabbit polyclonal antiserum (13) raised against homogeneous bovine DNA ligase I was added per ml of extract, and then the mixture was incubated at 0°C for 1 h. Immunocomplexes were affinity purified on 2% (wtlvol) protein A-Sepharose beads (4 Fast Flow; Pharmacia) at 4°C for 1 h with continuous mixing. The protein A-Sepharose beads were then washed three times with 1 ml of lysis buffer. Protein A-Sepharose-bound immunocomplexes containing DNA ligase I were used in in vitro DNA ligation assays. For immunoblotting analysis, DNA ligase I was releas...

CTG/CAG Repeat Instability Is Modulated by the Levels of Human DNA Ligase I and Its Interaction with Proliferating Cell Nuclear Antigen

“…In agreement with published studies, DNA replication efficiency was dependent upon having enough ligation activity and the ability of hLigI to interact with PCNA (16 -18, 33, 57-59). Expression of wild type hLigI mostly corrected the replication defect of hLigI-deficient 46BRLigI m/m extract (17,18,40). These results are also consistent with PCNA coordinating the action of hLigI during Okazaki fragment processing and ligation and the essential role of PCNA in efficient fork progression (17,40).…”

“…Expression of wild type hLigI mostly corrected the replication defect of hLigI-deficient 46BRLigI m/m extract (17,18,40). These results are also consistent with PCNA coordinating the action of hLigI during Okazaki fragment processing and ligation and the essential role of PCNA in efficient fork progression (17,40). At the same time, replication was slightly hindered by the presence of CTG/CAG repeats in the template, an effect further exacerbated by hLigI-altered backgrounds.…”

“…3). Anomalously long repair patch sizes have previously been observed in other studies for 46BR.1G1 but not with added functional hLigI (17,55). Such altered repair tract sizes for slipped-DNAs may reflect aberrant repair that may lead to instability.…”

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

“…This mutation does not affect adenylate complex formation but does affect nick ligation consistent with our model [26]. Notably, mice expressing this mutation exhibit an increased predisposition to cancer [27].…”

Interactions of the DNA Ligase IV-XRCC4 Complex with DNA Ends and the DNA-dependent Protein Kinase

“…The initial symptoms of the individual with DNA ligase I deficiency were recurrent infections caused by severe combined immunodeficiency (17). Cell lines established from this patient exhibited defects in Okazaki fragment joining and sensitivity to a wide range of DNA-damaging agents, a phenotype consistent with the predicted role of DNA ligase I in DNA replication and excision repair (18,19). When the DNA ligase I-deficient cells were activated for V(D)J recombination by ectopic expression of the RAG proteins, there was no apparent defect in this type of recombination (20,21).…”