DNA Ligase I, the Replicative DNA Ligase

Howes, Timothy; Tomkinson, Alan E.

doi:10.1007/978-94-007-4572-8_17

Cited by 56 publications

(38 citation statements)

References 51 publications

(101 reference statements)

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“…These primers must be removed before ligation of the Okazaki fragment to the lagging strand, because of their RNA content and also because Pol α does not possess a proofreading 3’ to 5’ exonuclease and is error prone. Thus, the process requires the removal of the RNA/DNA primer when it is encountered by a new Okazaki fragment and the formation of a nick (reviewed in [1–3]) that can be sealed by DNA ligase I [4,5]).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

et al. 2013

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Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5’-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.

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Section: Introductionmentioning

confidence: 99%

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

et al. 2013

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“…Although it appears that degradation of LigI is enhanced in non-dividing cells, further studies are needed to determine whether the ubiquitylation of LigI by the Cul4-DDB1-DCAF7 complex varies during the cell cycle and in response to inhibition of proliferation and whether cell cycledependent phosphorylation of LigI regulates its ubiquitylation (44,45) The efficient and accurate replication of DNA requires a high degree of coordination among a large number of proteins. This is particularly evident for lagging strand DNA synthesis where protein-protein interactions with PCNA coordinate the sequential actions of the enzymes that synthesize, process, and join Okazaki fragments (46). This mechanism, which involves transient interactions with overlapping binding sites on the PCNA trimer, is prone to disruption by changes in the relative stoichiometry of the PCNA-interacting proteins (12,13,18).…”

Section: Discussionmentioning

confidence: 99%

Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex

Peng

Liao

Matsumoto

et al. 2016

Journal of Biological Chemistry

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The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.A large number of single-strand interruptions are generated during DNA replication because of the discontinuous nature of lagging strand DNA synthesis. These unlinked Okazaki fragments are joined together by a DNA ligase to generate an intact strand. In humans, there are three genes that encode DNA ligases, LIG1, LIG3, and LIG4 (1-3). There is compelling cell biology, biochemical, and molecular genetic evidence indicating that the DNA ligase encoded by the mammalian LIG1 gene, DNA ligase I (LigI), 4 plays the predominant role in DNA replication. The expression of the LIG1 gene is increased when quiescent cells are induced to proliferate (4). In addition, LigI physically and functionally interacts with two key DNA replication protein complexes, proliferating cell nuclear antigen (PCNA) and replication factor C (RFC), and co-localizes with these factors in replication foci during S phase (5-8). Finally, the human ligI cell line 46BR.1G1 has a defect in joining Okazaki fragments that is complemented by the expression of wild-type LigI (6, 9).The 46BR.1G1 cell line was established from a patient with growth retardation, sunlight sensitivity, and severe immunodeficiency (10). This individual was a compound heterozygote with one allele (Glu-566 to Lys) encoding an inactive polypeptide and the other (Arg-771 to Trp) encoding a version of LigI with about 20-fo...

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“…The crystal structures also revealed conformational changes upon Pol β binding to DNA. The crystal structures of ligase I (for reviews see [4,20,21]) bound to DNA reveal those subdomains that encircle DNA and the position of the nicked DNA ends within the ligase active site. In addition to its repair function, ligase l functions during DNA replication of Okazaki fragments.…”

Section: Base Excision Repair Enzyme Structures Inform Functionmentioning

confidence: 99%

Base excision repair: A critical player in many games

2014

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This perspective reviews the many dimensions of base excision repair from a 10,000 foot vantage point and provides one person’s view on where the field is headed. Enzyme function is considered under the lens of X-ray diffraction and single molecule studies. Base excision repair in chromatin and telomeres, regulation of expression and the role of posttranslational modifications are also discussed in the context of enzyme activities, cellular localization and interacting partners. The specialized roles that base excision repair play in transcriptional activation by active demethylation and targeted oxidation as well as how base excision repair functions in the immune processes of somatic hypermutation and class switch recombination and its possible involvement in retroviral infection are also discussed. Finally the complexities of oxidative damage and its repair and its link to neurodegenerative disorders, as well as the role of base excision repair as a tumor suppressor are examined in the context of damage, repair and aging. By outlining the many base excision repair-related mysteries that have yet to be unraveled, hopefully this perspective will stimulate further interest in the field.

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DNA Ligase I, the Replicative DNA Ligase

Cited by 56 publications

References 51 publications

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex

Base excision repair: A critical player in many games

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