Sustained active site rigidity during synthesis by human DNA polymerase μ

Moon, A.F.; Pryor, John M.; Ramsden, Dale A.; Kunkel, Thomas A.; Bębenek, Katarzyna; Pedersen, Lars C.

doi:10.1038/nsmb.2766

Cited by 57 publications

(134 citation statements)

References 57 publications

Supporting

Mentioning

128

Contrasting

Order By: Relevance

“…2B). This is consistent with the apparent rigidity of the global protein fold observed for hPol μ Δ2 during catalysis on a 1-nt gapped DNA substrate (15). Comparison of the hPol μ Δ2 complexes with 1-and 2-nt gapped substrates similarly demonstrates minimal structural differences in the overall protein fold (rms deviations of less than 0.4 Å; Table S3).…”

Section: Discussionsupporting

confidence: 71%

“…The 3ʹ-OH lies 3.6 Å from the α-phosphate of the dUMPNPP (Fig. 3 A and B), a distance consistent with that observed in the 1-nt gapped ternary complex (15). As in the binary complex, the primer terminal sugar adopts a C3ʹ-endo sugar pucker and contributes to coordination of the ion in the metal A site.…”

Section: Resultssupporting

confidence: 61%

“…Interactions between the thumb and the template strand shape the nascent base-pair binding site. Similar to the structure of hPol μ Δ2 in binary complex with a 1-nt gapped DNA substrate (15), both the protein and the DNA are present in the "closed" conformation, with the primer terminus adopting a C3ʹ-endo sugar pucker (Fig. 2C).…”

Section: Resultsmentioning

confidence: 89%

“…2 A-C). This variant has enzymatic properties indistinguishable from those of the wildtype catalytic domain, and has a higher propensity to crystallize (15). Using hPol μ Δ2, the catalytic cycle of synthesis on a 2-nt gapped DNA substrate has been structurally characterized, from the binary complex [Protein Data Bank (PDB) ID code 4YCX] through nucleotide incorporation.…”

Section: Resultsmentioning

confidence: 99%

“…S1). Although there is currently no means of determining a causal relationship between the movement of loop 1 and that of the DNA template, it should be noted that no such movement occurs in loop 1 as a result of nucleotide incorporation for hPol μ Δ2 on a 1-nt gapped substrate (15). Movement of loop 1 in this particular context is likely to be a function of nucleotide incorporation, given that the position of the loop is not constrained by crystal-packing interactions in either the 1-or 2-nt gapped lattices, and might represent attempted translocation or dissociation.…”

Section: Minimal Structural Changes Occur As a Results Of Nucleotide Imentioning

confidence: 99%

See 4 more Smart Citations

Creative template-dependent synthesis by human polymerase mu

Moon

Gosavi

Kunkel

et al. 2015

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

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 71%

Section: Resultssupporting

confidence: 61%

Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Minimal Structural Changes Occur As a Results Of Nucleotide Imentioning

confidence: 99%

See 3 more Smart Citations

Creative template-dependent synthesis by human polymerase mu

Moon

Gosavi

Kunkel

et al. 2015

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

Self Cite

View full text Add to dashboard Cite

show abstract

Structure and function relationships in mammalian DNA polymerases

Hoitsma

Whitaker

Schaich

et al. 2019

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization. In this review, the structure and function of all 15 mammalian DNA polymerases from families B, Y, X, and A will be reviewed and discussed with special emphasis on the insights gleaned from recently published atomic resolution structures.

show abstract

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes

2018

View full text Add to dashboard Cite

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various subpathways. We highlight the differences between homologous recombination and end-joining mechanisms including non-homologous end-joining and microhomology-mediated end-joining and offer insights into how these pathways are regulated. Finally, we introduce noncanonical functions of the recombination proteins, in particular during DNA replication stress.

show abstract

Sustained active site rigidity during synthesis by human DNA polymerase μ

Cited by 57 publications

References 57 publications

Creative template-dependent synthesis by human polymerase mu

Creative template-dependent synthesis by human polymerase mu

Structure and function relationships in mammalian DNA polymerases

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes

Contact Info

Product

Resources

About