Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase μ at an NHEJ double-strand break junction

Loc'h, J.; Gerodimos, C.A.; Rosario, Sandrine; Tekpinar, Mustafa; Lieber, Michael R.; Delarue, Marc

doi:10.1074/jbc.ra119.008739

Cited by 10 publications

(9 citation statements)

References 59 publications

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“…The "wedging" phenomenon is not observed in the hPolμΔ2 complementary DSB substrate. A recent study of a chimeric form of mouse TdT harboring the Loop1 region from mouse Polμ (henceforth, referred to as TdT-μ) included crystal structures of the chimera in complex with a 1ntgapped SSB substrate (PDB ID code 6GO5 24 , Fig. 7a, b) and with a noncomplementary DSB synaptic complex reinforced by a triplex-forming oligonucleotide (PDB ID code 6GO7 24 , Fig.…”

Section: Discussionmentioning

confidence: 99%

“… Superposition of hPolμΔ2 complementary DSB (colored as in Fig. 1a–b ) with mouse TdT a 1nt-gapped SSB (light pink, PDB ID code 6GO5 24 ) or c synapse with noncomplementary DSB (light gray, PDB ID code 6GO7 24 ) substrates, with zoomed-in view of superimposed DNA substrates b , d . e Superimposed DNA substrates from the chimeric TdT-μ crystal structures, colored as in c , d , to highlight the “wedged” upstream residues (dashed yellow circle).…”

Section: Discussionmentioning

confidence: 99%

“…Though there exists a plethora of biochemical [13][14][15][16] and structural [17][18][19][20][21] information illustrating the activity of these Family X polymerases on single-strand break (SSB) substrates, understanding how each individual polymerase copes with DSB end-bridging is unclear. Thus far, only DSBbound crystal structures of murine TdT 22,23 and a chimeric construct of murine TdT containing the Loop1 region from murine Polμ 24 are currently available, providing an important yet incomplete portrait of Family X polymerase behavior during NHEJ. We therefore present high-resolution crystal structures of the human Polμ catalytic domain simultaneously engaging both ends of a complementary DSB substrate.…”

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confidence: 99%

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Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

et al. 2020

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Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

et al. 2020

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“…Compared with replicative DNA polymerase, Pol μ lacks proofreading activity, with an intrinsically high error rate of 10 −3 –10 −5 for dNTP incorporation 16 , 17 . Despite its reduced fidelity, Pol μ containing an insertion in Loop1 can fill small gapped DNA substrates 18 – 21 , and a recent study revealed that Pol μ can effectively misinsert dGTP opposite 1-nt gapped DNA containing templating T 22 . This misincorporation further facilitates subsequent NHEJ ligation reaction by the DNA ligase IV/XRCC4 complex, which may ultimately lead to genomic instability during NHEJ repair.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation

et al. 2021

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Pol μ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol μ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol μ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol μ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3ʹ end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol μ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.

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“…7 Switching of Loop1 between TdT and Pol μ led to partial alteration of DNA substrate preference. 8,9 With TdT's unique function, it is widely used in the clinical and biotechnology field. One of the most common uses of TdT is in the TUNEL.…”

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confidence: 99%

Evolving a Thermostable Terminal Deoxynucleotidyl Transferase

Chua

Osothprarop

et al. 2020

ACS Synth. Biol.

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Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of a 3′ reversibly blocked nucleotide, followed by deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3′ modified nucleotides, and TdT engineering is hampered by the fact that TdT is marginally stable and only present in mesophilic organisms. We sought to first evolve a thermostable TdT variant to serve as backbone for subsequent evolution to enable efficient incorporation of 3′-modified nucleotides. A thermostable variant would be a good starting point for such an effort, as evolution to incorporate bulky modified nucleotides generally results in lowered stability. In addition, a thermostable TdT would also be useful when blunt dsDNA is a substrate as higher temperature could be used to melt dsDNA. Here, we developed an assay to identify thermostable TdT variants. After screening about 10 000 TdT mutants, we identified a variant, named TdT3-2, that is 10 °C more thermostable than WT TdT, while preserving the catalytic properties of the WT enzyme.

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Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase μ at an NHEJ double-strand break junction

Cited by 10 publications

References 59 publications

Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation

Evolving a Thermostable Terminal Deoxynucleotidyl Transferase

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