Mammalian Dna Ligases

Lindahl, Tomas; Barnes, Deborah E.

doi:10.1146/annurev.bi.61.070192.001343

Cited by 206 publications

(109 citation statements)

References 33 publications

(44 reference statements)

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“…2), mtDNA ligase (12), and DNA ligase I (data not shown). Because ATPdependent DNA ligases as a class share structural and functional features (22), it is likely that the presence of AP lyase activity will be conserved in this family. To date we have not been able to document AP lyase activity in bacterial DNA ligases from either E. coli or T. aquaticus either in the presence or the absence of their cofactor, NAD (data not shown).…”

Section: Resultsmentioning

confidence: 99%

The Action of DNA Ligase at Abasic Sites in DNA

Bogenhagen

Pinz

1998

Journal of Biological Chemistry

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Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5-dRP group may be removed by AP lyase enzymes that employ a ␤-elimination mechanism. This ␤-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA.DNA repair pathways have evolved to process a wide range of chemically distinct lesions in DNA (1). One of the most common types of damage is spontaneous or enzymatic hydrolysis of the N-glycosidic bond between a DNA base and the sugar phosphate backbone generating an abasic site (AP site). 1 AP sites are highly mutagenic and require rapid and efficient repair. AP sites are processed by a base excision repair pathway that is frequently initiated by the action of a class II AP endonuclease that cleaves the DNA backbone adjacent to the lesion to produce 3Ј-OH and 2Ј-deoxyribose 5Ј-phosphate termini (2). The latter residue, referred to as a 5Ј-dRP moiety, is relatively alkali labile and can be removed by AP lyases that facilitate ␤-elimination. Most enzymes demonstrated to have dRPase activity operate through this lyase mechanism, although some, such as the Escherichia coli recJ protein, catalyze hydrolysis (3). The two classes of dRPase enzymes can be distinguished by the fact that the lyase mechanism frequently involves formation of a transient Schiff base intermediate in which an amino group on the enzyme is covalently bound to the DNA. This lyase mechanism, first proposed for E. coli endonuclease III (4), provides a simple method to identify a polypeptide with AP lyase activity because the Schiff base intermediate can be trapped in a stable form by reaction with a strong reducing agent, such as NaBH 4 or NaBH 3 CN. Thus, with an appropriate radioactively labeled substrate, the label can be transferred to the AP lyase enzyme. This borohydride trapping has been documented for several repair enzymes (5-9) and, more recently, for DNA pol ␤ (10, 11).The combined action of AP endonuclease and AP lyase leaves a one-nucleotide gap that is filled by a DNA polymerase. The final step in repair involves DNA strand sealing by DNA ligase. The mechanism of DNA ligase involves covalent modification of the enzyme by adenylation, transfer of the AMP residue in a phosphoanhydride linkage to the ...

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Section: Resultsmentioning

confidence: 99%

The Action of DNA Ligase at Abasic Sites in DNA

Bogenhagen

Pinz

1998

Journal of Biological Chemistry

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“…SV40 large T antigen (TAg) 1 was obtained from Molecular Biology Resources, Inc. (Milwaukee, WI), and T4 DNA ligase was from New England Biolabs Inc. Recombinant human DNA ligase III and XRCC1 proteins were produced as described (15).…”

Section: Reagent Enzymesmentioning

confidence: 99%

“…Several distinct DNA ligases have been identified in mammalian cells, DNA ligase I being a major activity in proliferating cells (1,2). Cytostaining experiments with antibodies against DNA ligase I showed that the enzyme is specifically localized in the nucleus with the same granular staining pattern as DNA polymerase ␣, implicating DNA ligase I in DNA replication (3).…”

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confidence: 99%

Specific Function of DNA Ligase I in Simian Virus 40 DNA Replication by Human Cell-free Extracts Is Mediated by the Amino-terminal Non-catalytic Domain

Mackenney¹,

Barnes²,

Lindahl³

1997

Journal of Biological Chemistry

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The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I. This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro. Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable. Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts. The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the fulllength protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication. Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain. This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.Several distinct DNA ligases have been identified in mammalian cells, DNA ligase I being a major activity in proliferating cells (1, 2). Cytostaining experiments with antibodies against DNA ligase I showed that the enzyme is specifically localized in the nucleus with the same granular staining pattern as DNA polymerase ␣, implicating DNA ligase I in DNA replication (3). DNA ligase I (in conjunction with DNA polymerase ␣, ␦, or ⑀; RNase H1; and the 5Ј-nuclease DNase IV/ FEN-1) is able to complete lagging strand DNA replication in vitro on a synthetic DNA substrate (4), and DNA ligase I activity also functions to generate closed circular DNA during SV40 DNA replication reconstituted with SV40 large T antigen and purified mammalian proteins (5, 6).The human DNA ligase I cDNA encodes a 102-kDa polypeptide (7). A 78-kDa carboxyl-terminal domain shows significant amino acid sequence homology to the CDC9 and cdc17 ϩ gene products of Saccharomyces cerevisiae and Schizosaccharomyces pombe as well as to the human DNA ligase III and IV cDNAs. This domain is catalytically active in vitro in the absence of the amino-terminal region and is able to complement a conditional/ lethal DNA ligase mutant of Escherichia coli (8). The 24-kDa amino-terminal portion of human DNA ligase I is a proteasesensitive hydrophilic region that has no counterpart in other mammalian DNA ligases or the yeast DNA ligases. Although this amino-terminal region is not required for activity of DNA ligase I in standard in vitro DNA joining assays, it is essential in vivo to counteract the lethal effect of knocking out DNA ligase I in mouse embryonic stem cells by the ectopic expression of DNA ligase I (9). A functional role for the amino-terminal portion of DNA ligase I during DNA replication has not been directly demonstrated, but this region may serve to locali...

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“…The ATP-dependent DNA ligases catalyze the joining of 5′-phosphate-terminated strands to 3′-hydroxyl-terminated strands via three sequential nucleotidyl transfer reactions (1)(2)(3). In the first step, attack on the α-phosphate of ATP by DNA ligase results in displacement of pyrophosphate and formation of a covalent ligase-adenylate intermediate in which AMP is linked to the ε-amino group of a lysine.…”

Section: Introductionmentioning

confidence: 99%