Last Stop on the Road to Repair: Structure of E. coli DNA Ligase Bound to Nicked DNA-Adenylate

Nandakumar, Jayakrishnan; Nair, Pravin A.; Shuman, Stewart

doi:10.1016/j.molcel.2007.02.026

Cited by 88 publications

(146 citation statements)

References 60 publications

(86 reference statements)

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“…Importantly, the adjacent configuration is consistent with an interaction between XRCC4/ XLF/LigIV proteins on each dsDNA strand that is localized away from the ends, as would be anticipated in filaments. Crystal structures of ligases interacting with strand-break substrates indicate that an end-to-end configuration is required for the final step in NHEJ (51,52). We suggest that the alternate, adjacent configuration that we observed here is critical to allow engagement of ends by processing enzymes (e.g., Artemis).…”

Section: Discussionmentioning

confidence: 61%

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Reid

Keegan

Leo‐Macias

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

154

168

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Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by singlemolecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.genomic integrity | DNA repair | nonhomologous end-joining | super-resolution microscopy | single-molecule FRET C hromosomal double-strand breaks (DSBs), considered the most cytotoxic form of DNA damage, occur as a result of normal cellular processes (1, 2) as well as cancer therapies (3-5). The cellular DNA damage response (DDR) and repair pathways responsible for maintaining genomic integrity are highly regulated and synchronized processes, both temporally and spatially, involving the coordinated recruitment, assembly, and disassembly of numerous macromolecular complexes (6, 7). In mammalian cells, nonhomologous end-joining (NHEJ) is the primary DSB repair pathway; it is active throughout the cell cycle and is crucial for viability. Dysfunctional NHEJ is associated with several clinical conditions, including LIG4 syndrome and severe combined immunodeficiency (1,8). Despite its importance, however, the details of how the NHEJ complex assembles at DSBs, brings together a pair of breaks, and organizes subsequent catalytic repair steps remain unknown.In NHEJ, DSBs are initially recognized by the Ku 70/80 heterodimer (Ku), which encircles dsDNA ends (Ku:DNA) and serves as a molecular scaffold for recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4 like factor), and DNA ligase IV (LigIV) (1,(9)(10)(11)(12)(13)(14). Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15-18); however, recent experiments indicate that DNAPKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered . In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26-28). Whether such filaments mediate repair in vivo has not yet been determined.Our present understanding of the cellular NHEJ response to DSBs ...

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Section: Discussionmentioning

confidence: 61%

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Reid

Keegan

Leo‐Macias

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

154

168

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“…However, the identification of discrete metalbinding sites within DNA ligase structures has been elusive. Crystal structures of viral, bacterial, and mammalian DNA ligases include extra electron density within the active site that has been interpreted as bound metal ion(s) or a bound water standing in for a metal that is coordinated by protein oxygen ligands (16,18,(25)(26)(27). There is uncertainty about the number of metals and their locations during each step of end joining because of the limited quality of electron density, particularly when using anomalous X-ray scattering to locate metal-binding sites.…”

Section: Structures Of Eukaryotic Dna Ligasesmentioning

confidence: 99%

Eukaryotic DNA Ligases: Structural and Functional Insights

Ellenberger

Tomkinson

2008

Annu. Rev. Biochem.

294

338

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DNA ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA ligases. Members of the DNA ligase I and IV families are found in all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.

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“…The DBD of LigI was shown to be essential for DNA nick joining activity, and it makes the greatest contribution of any domain to DNA binding affinity (18). DNA ligases of bacteria and viruses also appear to be capable of wrapping around DNA (18,20). Escherichia coli LigA and related NAD-dependent ligases have a C-terminal helix-hairpin-helix domain that functions similarly to the N-terminal DBD of LigI (20,21).…”

mentioning

confidence: 99%

“…DNA ligases of bacteria and viruses also appear to be capable of wrapping around DNA (18,20). Escherichia coli LigA and related NAD-dependent ligases have a C-terminal helix-hairpin-helix domain that functions similarly to the N-terminal DBD of LigI (20,21). Even Chlorella virus ligase, which is the smallest known eukaryotic DNA ligase and lacks a DBD, has a small latch structure that allows the enzyme to fully encircle the DNA (22).…”

mentioning

confidence: 99%