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DNA-binding determinants promoting NHEJ by human Polμ.

Martin MJ, Juarez R, Blanco L - Nucleic Acids Res. (2012)

Bottom Line: Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding.Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction.Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Dynamics and Function, Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain.

ABSTRACT
Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

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Impact of Polµ DNA-binding properties on its enzymatic activity during NHEJ. (A) Gap-filling activity of Polβ, Polλ and Polµ (25 nM each) was assayed using a substrate formed by the hybridization of the oligonucleotides SP1C, T28 and D12. When indicated, 10 nM of each dNTP was added, in the presence of 2.5 mM MgCl2. (B) NHEJ assay of Polβ (600 nM), Polλ (600 nM) and Polµ (200 nM) was performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (shown in light gray) and the cold substrate by hybridization of CA and NHEJ-D (shown in dark gray). When indicated, dNTPs were added separately at 100 µM in the presence of 1 mM MnCl2 for Polβ and Polλ, and 2.5 mM MgCl2 for Polµ. After electrophoresis, the labeled fragments were detected by autoradiography. (C) NHEJ reaction performed as in (B), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C- and D-NHEJ. When indicated, the substrates contain a 5′-P group at the downstream strand (dark gray spheres).
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gks896-F4: Impact of Polµ DNA-binding properties on its enzymatic activity during NHEJ. (A) Gap-filling activity of Polβ, Polλ and Polµ (25 nM each) was assayed using a substrate formed by the hybridization of the oligonucleotides SP1C, T28 and D12. When indicated, 10 nM of each dNTP was added, in the presence of 2.5 mM MgCl2. (B) NHEJ assay of Polβ (600 nM), Polλ (600 nM) and Polµ (200 nM) was performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (shown in light gray) and the cold substrate by hybridization of CA and NHEJ-D (shown in dark gray). When indicated, dNTPs were added separately at 100 µM in the presence of 1 mM MnCl2 for Polβ and Polλ, and 2.5 mM MgCl2 for Polµ. After electrophoresis, the labeled fragments were detected by autoradiography. (C) NHEJ reaction performed as in (B), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C- and D-NHEJ. When indicated, the substrates contain a 5′-P group at the downstream strand (dark gray spheres).

Mentions: In agreement with their similar Polβ-like core, the three template-directed polymerases from the human X family, i.e. Polβ, Polλ and Polµ, are similarly competent when polymerizing on a small gap (as shown in Figure 4A). Conversely, it has been shown that Polβ is not a player of the NHEJ pathway, while Polλ and Polµ, having a BRCT domain that interacts with NHEJ core factors, are the main polymerases implicated; moreover, Polλ and Polµ are not redundant since in the presence of NHEJ accessory factors such as Ku70/80 and XRCC4/LigaseIV, both of them can use complementary ends but only Polµ is able to join non-complementary ends (11). To evaluate if these differences are intrinsic to each enzyme, we tested the three polymerases Polβ, Polλ and Polµ, in NHEJ in vitro reactions in which the polymerase alone, in the absence of NHEJ accessory factors, is challenged to bridge two DNA ends, extending one 3′-end by copying the templating base provided by a second 3′-end. For that we used two sets of short double-stranded DNA molecules: one set containing a 3′-overhang of 2 nt which can form 1 bp (Figure 4B, scheme), and the other set of DNA ends, whose 1-nt 3′-protrusion (dC) provides complementarity when confronted to each other (Figure 4C, scheme). In the two cases, one DNA end (depicted in light gray) was labeled at the 5′-terminus of the 3′-protruding strand, and therefore can be assayed for primer extension, whereas the second molecule (depicted in dark gray), unlabeled, provides the templating base (dC) in trans. Thus, preferential extension of the primer with ddG (having the same affinity as dG in all PolXs, but used to avoid any extension further to +1) would be indicative of an accurate first stage of NHEJ. When working with DNA substrates bearing complementary 3′-protruding ends, we observed that, as expected, Polβ had very little activity, while Polλ and Polµ can efficiently and accurately insert the correct nucleotide (ddG) dictated by the templating base (dC) provided in trans by the second end (Figure 4B). On the other hand, when using DNA substrates that did not contain any microhomology, both Polβ and Polλ were inactive and only Polµ was able to perform an efficient and accurate NHEJ reaction (Figure 4C), a similar behavior shown to occur at some incompatible ends when Polµ was assisted by NHEJ core factors (29). Therefore, our results allow to conclude that the capacity of Polµ to perform NHEJ of incompatible ends is an intrinsic property of the enzyme, not conferred by additional NHEJ protein factors.Figure 4.


DNA-binding determinants promoting NHEJ by human Polμ.

Martin MJ, Juarez R, Blanco L - Nucleic Acids Res. (2012)

Impact of Polµ DNA-binding properties on its enzymatic activity during NHEJ. (A) Gap-filling activity of Polβ, Polλ and Polµ (25 nM each) was assayed using a substrate formed by the hybridization of the oligonucleotides SP1C, T28 and D12. When indicated, 10 nM of each dNTP was added, in the presence of 2.5 mM MgCl2. (B) NHEJ assay of Polβ (600 nM), Polλ (600 nM) and Polµ (200 nM) was performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (shown in light gray) and the cold substrate by hybridization of CA and NHEJ-D (shown in dark gray). When indicated, dNTPs were added separately at 100 µM in the presence of 1 mM MnCl2 for Polβ and Polλ, and 2.5 mM MgCl2 for Polµ. After electrophoresis, the labeled fragments were detected by autoradiography. (C) NHEJ reaction performed as in (B), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C- and D-NHEJ. When indicated, the substrates contain a 5′-P group at the downstream strand (dark gray spheres).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526283&req=5

gks896-F4: Impact of Polµ DNA-binding properties on its enzymatic activity during NHEJ. (A) Gap-filling activity of Polβ, Polλ and Polµ (25 nM each) was assayed using a substrate formed by the hybridization of the oligonucleotides SP1C, T28 and D12. When indicated, 10 nM of each dNTP was added, in the presence of 2.5 mM MgCl2. (B) NHEJ assay of Polβ (600 nM), Polλ (600 nM) and Polµ (200 nM) was performed as described in ‘Materials and Methods’ section, using a set of compatible substrates: the labeled substrate was formed by hybridization of GT and NHEJ-D (shown in light gray) and the cold substrate by hybridization of CA and NHEJ-D (shown in dark gray). When indicated, dNTPs were added separately at 100 µM in the presence of 1 mM MnCl2 for Polβ and Polλ, and 2.5 mM MgCl2 for Polµ. After electrophoresis, the labeled fragments were detected by autoradiography. (C) NHEJ reaction performed as in (B), with a set of incompatible substrates in which both the labeled (light gray) and cold (dark gray) molecules were formed by hybridization of C- and D-NHEJ. When indicated, the substrates contain a 5′-P group at the downstream strand (dark gray spheres).
Mentions: In agreement with their similar Polβ-like core, the three template-directed polymerases from the human X family, i.e. Polβ, Polλ and Polµ, are similarly competent when polymerizing on a small gap (as shown in Figure 4A). Conversely, it has been shown that Polβ is not a player of the NHEJ pathway, while Polλ and Polµ, having a BRCT domain that interacts with NHEJ core factors, are the main polymerases implicated; moreover, Polλ and Polµ are not redundant since in the presence of NHEJ accessory factors such as Ku70/80 and XRCC4/LigaseIV, both of them can use complementary ends but only Polµ is able to join non-complementary ends (11). To evaluate if these differences are intrinsic to each enzyme, we tested the three polymerases Polβ, Polλ and Polµ, in NHEJ in vitro reactions in which the polymerase alone, in the absence of NHEJ accessory factors, is challenged to bridge two DNA ends, extending one 3′-end by copying the templating base provided by a second 3′-end. For that we used two sets of short double-stranded DNA molecules: one set containing a 3′-overhang of 2 nt which can form 1 bp (Figure 4B, scheme), and the other set of DNA ends, whose 1-nt 3′-protrusion (dC) provides complementarity when confronted to each other (Figure 4C, scheme). In the two cases, one DNA end (depicted in light gray) was labeled at the 5′-terminus of the 3′-protruding strand, and therefore can be assayed for primer extension, whereas the second molecule (depicted in dark gray), unlabeled, provides the templating base (dC) in trans. Thus, preferential extension of the primer with ddG (having the same affinity as dG in all PolXs, but used to avoid any extension further to +1) would be indicative of an accurate first stage of NHEJ. When working with DNA substrates bearing complementary 3′-protruding ends, we observed that, as expected, Polβ had very little activity, while Polλ and Polµ can efficiently and accurately insert the correct nucleotide (ddG) dictated by the templating base (dC) provided in trans by the second end (Figure 4B). On the other hand, when using DNA substrates that did not contain any microhomology, both Polβ and Polλ were inactive and only Polµ was able to perform an efficient and accurate NHEJ reaction (Figure 4C), a similar behavior shown to occur at some incompatible ends when Polµ was assisted by NHEJ core factors (29). Therefore, our results allow to conclude that the capacity of Polµ to perform NHEJ of incompatible ends is an intrinsic property of the enzyme, not conferred by additional NHEJ protein factors.Figure 4.

Bottom Line: Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding.Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction.Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Genome Dynamics and Function, Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain.

ABSTRACT
Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5'-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5'-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys(249), Arg(253) and Arg(416)) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5'-P, thus boosting Polµ-mediated NHEJ reactions.

Show MeSH
Related in: MedlinePlus