The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining

Waters, Crystal A.; Strande, Natasha T.; Pryor, John M.; Christina, Strom,; Mieczkowski, Piotr A.; Burkhalter, Martin D.; Oh, Sehyun; Qaqish, Bahjat F.; Moore, Dominic T.; Hendrickson, Eric A.; Ramsden, Dale A.

doi:10.1038/ncomms5286

Cited by 75 publications

(64 citation statements)

References 58 publications

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“…The ligation step during NHEJ is much less tolerant of mispairs and other distortions when these are 3′ of strand breaks, relative to 5′ of strand breaks (2). As a consequence, synthesis that initiates from 3′ overhangs or blunt ends-and that then is sufficient to generate paired termini 3′ of strand breaks-is especially significant to this pathway.…”

Section: Discussionmentioning

confidence: 99%

“…This assay has previously been validated as efficient, reproducible, and linear over the range relevant to these experiments (2). The number of junctions recovered from cells was determined by comparison of the threshold cycles (C T ) to a standard curve run in parallel, generated by serial dilution of a model amplicon into DNA harvested from mocktransfected DNA.…”

Section: Methodsmentioning

confidence: 99%

“…This pathway is thus essential for assembly of the antigen receptor genes (Ig and TcR) required for adaptive immunity, normal cellular replicative capacity and nervous system development (1) and shapes the effectiveness and safety of break-inducing cancer therapies (ionizing radiation and certain chemotherapeutic agents) (2). Repair is accomplished by directly ligating broken ends together.…”

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confidence: 99%

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Essential role for polymerase specialization in cellular nonhomologous end joining

Pryor

Waters

Aza

et al. 2015

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

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Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) μ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol μ using the unpaired base adjacent to the downstream 5′ phosphate even when there are available template sites further upstream of this position; this makes Pol μ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol μ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.nonhomologous end joining | double-strand break repair | Pol X | polymerase mu | polymerase lambda

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Essential role for polymerase specialization in cellular nonhomologous end joining

Pryor

Waters

Aza

et al. 2015

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

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“…Two DNA ends must be closely juxtaposed for ligation to occur, although emerging data suggest that repair of the two strands occurs independently (12,46,47). It is also well appreciated that DSBs can be maintained in a spatiotemporal manner in large DNA repair centers (48).…”

Section: Xlfmentioning

confidence: 99%

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

Roy

Melo

et al. 2015

Molecular and Cellular Biology

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fThe classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA ends in vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNA in vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4 in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF-or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.

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“…DSB substrates lacking microhomology at the break site are unsuitable for immediate rejoining by ligase IV, and a polymerase is usually required to fill the gaps to generate ends that can be efficiently ligated (3). In higher eukaryotes, this role is performed by family X polymerase λ (Pol λ) and polymerase μ (Pol μ) (4)(5)(6).…”

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Creative template-dependent synthesis by human polymerase mu

Moon

Gosavi

Kunkel

et al. 2015

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

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Among the many proteins used to repair DNA double-strand breaks by nonhomologous end joining (NHEJ) are two related family X DNA polymerases, Pol λ and Pol μ. Which of these two polymerases is preferentially used for filling DNA gaps during NHEJ partly depends on sequence complementarity at the break, with Pol λ and Pol μ repairing complementary and noncomplementary ends, respectively. To better understand these substrate preferences, we present crystal structures of Pol μ on a 2-nt gapped DNA substrate, representing three steps of the catalytic cycle. In striking contrast to Pol λ, Pol μ "skips" the first available template nucleotide, instead using the template base at the 5′ end of the gap to direct nucleotide binding and incorporation. This remarkable divergence from canonical 3′-end gap filling is consistent with data on end-joining substrate specificity in cells, and provides insights into polymerase substrate choices during NHEJ.DNA polymerase mu | DNA polymerase lambda | DNA repair | nonhomologous end joining D NA double-strand breaks (DSBs) result from exposure to endogenous and exogenous factors including reactive oxygen species, physical or mechanical stress, ionizing radiation, or activities of nuclear enzymes on DNA (1). Another source of DSBs is programmed DNA breakage during meiotic or mitotic recombination, immunoglobin gene rearrangement in V(D)J, and class-switch recombination (1, 2). Because of the largely random nature of accidental double-strand breakage, the broken ends can have widely varying sequences, structures, or modifications. Nonhomologous end joining (NHEJ) is the predominant form of DSB repair in higher eukaryotes, and requires a certain degree of flexibility from the many factors involved in this complex repair process.DSB substrates lacking microhomology at the break site are unsuitable for immediate rejoining by ligase IV, and a polymerase is usually required to fill the gaps to generate ends that can be efficiently ligated (3). In higher eukaryotes, this role is performed by family X polymerase λ (Pol λ) and polymerase μ (Pol μ) (4-6). Pol λ has a strong preference for substrates with complementary template-strand pairing opposite the primer terminus. Pol μ can also use such complementary DSB substrates, but is uniquely active in template-dependent synthesis on DSBs entirely lacking complementarity, where the primer terminus is unpaired (7).Given partial overlap in substrate use in vitro by Pol μ and Pol λ, how does the NHEJ machinery select which enzyme to repair specific substrates with the highest fidelity? Recent work by Pryor et al. (8) (companion article in this issue) has demonstrated a strong preference for Pol λ over Pol μ in repairing the majority of complementary DSBs. This is advantageous, because Pol λ displays higher fidelity of synthesis (9, 10). However, Pol λ cannot use noncomplementary DSB substrates with unpaired primer termini. Pol μ is the only known polymerase that can use noncomplementary substrates, such that in Pol μ knockout cells, these ends are larg...

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The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining

Cited by 75 publications

References 58 publications

Essential role for polymerase specialization in cellular nonhomologous end joining

Essential role for polymerase specialization in cellular nonhomologous end joining

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

Creative template-dependent synthesis by human polymerase mu

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