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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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Ternary complex formation in solution. (A) Structure of Polµ (shown in wheat-colored ribbons) ternary complex (2IHM), in which the DNA substrate and incoming nucleotide are shown in sticks with the following colors: dNTP, dark teal; template strand, green; primer strand, yellow; downstream strand, dark pink. Selected residues are shown in red sticks. Nucleotides in the template strand are numbered as in the footprinting assays for clarity. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and Polµ (1.5 µg). When indicated, 100 µM dATP or dTTP were added, together with 2.5 mM MgCl2. The DNA substrate used, formed by hybridizing the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) always contains a phosphate group at the 5′-end of the downstream strand. Gel was dried and the labeled fragments detected by autoradiography. (C) Footprinting assays of Polµ mutants H329G and R387K (1.5 µg) were carried out as described in (B).
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gks896-F2: Ternary complex formation in solution. (A) Structure of Polµ (shown in wheat-colored ribbons) ternary complex (2IHM), in which the DNA substrate and incoming nucleotide are shown in sticks with the following colors: dNTP, dark teal; template strand, green; primer strand, yellow; downstream strand, dark pink. Selected residues are shown in red sticks. Nucleotides in the template strand are numbered as in the footprinting assays for clarity. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and Polµ (1.5 µg). When indicated, 100 µM dATP or dTTP were added, together with 2.5 mM MgCl2. The DNA substrate used, formed by hybridizing the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) always contains a phosphate group at the 5′-end of the downstream strand. Gel was dried and the labeled fragments detected by autoradiography. (C) Footprinting assays of Polµ mutants H329G and R387K (1.5 µg) were carried out as described in (B).

Mentions: In the case of Polβ, addition of the complementary deoxynucleotide (dT), but not the non-complementary (dA), enhanced the footprint of the polymerase (see a lighter band at position 37) and expanded further into the primer zone (Figure 2B, compare lanes 2–4 up to position 33), indicative of a subtle adjustment of the polymerase conformation, allowing a stronger binding to the primer region. Upon addition of either deoxynucleotide, a new hypersensitivity appears at position 29. It should be noted that there was an initial hypersensitivity at position 30, i.e. frequent cuts or nicks are produced by DNAseI at this position in the DNA substrate. These nicks are an ideal substrate for Polβ, since they mimic the intermediate substrates of the BER pathway in which this polymerase is implicated. As the footprint is being carried out in the presence of activating metal ions (Mg2+), Polβ would insert a deoxynucleotide on this nicked substrate, leading to the formation of a +1 product that appears at position 29. Changes at other bands can be also explained as polymerization events, not related to the specific footprint centered at the gap (position 41).Figure 2.


DNA-binding determinants promoting NHEJ by human Polμ.

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

Ternary complex formation in solution. (A) Structure of Polµ (shown in wheat-colored ribbons) ternary complex (2IHM), in which the DNA substrate and incoming nucleotide are shown in sticks with the following colors: dNTP, dark teal; template strand, green; primer strand, yellow; downstream strand, dark pink. Selected residues are shown in red sticks. Nucleotides in the template strand are numbered as in the footprinting assays for clarity. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and Polµ (1.5 µg). When indicated, 100 µM dATP or dTTP were added, together with 2.5 mM MgCl2. The DNA substrate used, formed by hybridizing the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) always contains a phosphate group at the 5′-end of the downstream strand. Gel was dried and the labeled fragments detected by autoradiography. (C) Footprinting assays of Polµ mutants H329G and R387K (1.5 µg) were carried out as described in (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks896-F2: Ternary complex formation in solution. (A) Structure of Polµ (shown in wheat-colored ribbons) ternary complex (2IHM), in which the DNA substrate and incoming nucleotide are shown in sticks with the following colors: dNTP, dark teal; template strand, green; primer strand, yellow; downstream strand, dark pink. Selected residues are shown in red sticks. Nucleotides in the template strand are numbered as in the footprinting assays for clarity. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and Polµ (1.5 µg). When indicated, 100 µM dATP or dTTP were added, together with 2.5 mM MgCl2. The DNA substrate used, formed by hybridizing the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) always contains a phosphate group at the 5′-end of the downstream strand. Gel was dried and the labeled fragments detected by autoradiography. (C) Footprinting assays of Polµ mutants H329G and R387K (1.5 µg) were carried out as described in (B).
Mentions: In the case of Polβ, addition of the complementary deoxynucleotide (dT), but not the non-complementary (dA), enhanced the footprint of the polymerase (see a lighter band at position 37) and expanded further into the primer zone (Figure 2B, compare lanes 2–4 up to position 33), indicative of a subtle adjustment of the polymerase conformation, allowing a stronger binding to the primer region. Upon addition of either deoxynucleotide, a new hypersensitivity appears at position 29. It should be noted that there was an initial hypersensitivity at position 30, i.e. frequent cuts or nicks are produced by DNAseI at this position in the DNA substrate. These nicks are an ideal substrate for Polβ, since they mimic the intermediate substrates of the BER pathway in which this polymerase is implicated. As the footprint is being carried out in the presence of activating metal ions (Mg2+), Polβ would insert a deoxynucleotide on this nicked substrate, leading to the formation of a +1 product that appears at position 29. Changes at other bands can be also explained as polymerization events, not related to the specific footprint centered at the gap (position 41).Figure 2.

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