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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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Related in: MedlinePlus

The BRCT domain of Polµ contributes, via DNA binding, to its intrinsic ability of joining DNA ends. (A) Gap-filling reactions were performed as described in ‘Materials and Methods’ section for the indicated proteins (25 nM) using a gapped substrate containing the oligonucleotides SP1C, T28 and D12. When indicated, dNTPs were added separately at 10 nM in the presence of 2.5 mM MgCl2. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and mutant and wild-type Polµ (1.5 µg). The DNA substrate used, formed by hybridization of the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) may have (P) or lack (OH) a phosphate group at the 5′-end of the downstream strand. (C) NHEJ reactions were performed as described in ‘Materials and Methods’ section, with 200 nM Polµ and using two sets of substrates: the labeled substrates were formed by hybridization of GT or C with NHEJ-D, and the cold substrates, by hybridization of either CA or C with NHEJ-D. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 10 µM in the presence of 2.5 mM MgCl2. The efficiency of the polymerization reaction is defined as a function of the percentage of primer extension. We used two different software programs to obtain the measurement of the extended primer in a minimum of three different replica experiments. (D) EMSA was performed with the indicated proteins (300 and 600 nM) using a 3′-protruding substrate containing the oligonucleotides GT and NHEJ-D.
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gks896-F8: The BRCT domain of Polµ contributes, via DNA binding, to its intrinsic ability of joining DNA ends. (A) Gap-filling reactions were performed as described in ‘Materials and Methods’ section for the indicated proteins (25 nM) using a gapped substrate containing the oligonucleotides SP1C, T28 and D12. When indicated, dNTPs were added separately at 10 nM in the presence of 2.5 mM MgCl2. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and mutant and wild-type Polµ (1.5 µg). The DNA substrate used, formed by hybridization of the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) may have (P) or lack (OH) a phosphate group at the 5′-end of the downstream strand. (C) NHEJ reactions were performed as described in ‘Materials and Methods’ section, with 200 nM Polµ and using two sets of substrates: the labeled substrates were formed by hybridization of GT or C with NHEJ-D, and the cold substrates, by hybridization of either CA or C with NHEJ-D. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 10 µM in the presence of 2.5 mM MgCl2. The efficiency of the polymerization reaction is defined as a function of the percentage of primer extension. We used two different software programs to obtain the measurement of the extended primer in a minimum of three different replica experiments. (D) EMSA was performed with the indicated proteins (300 and 600 nM) using a 3′-protruding substrate containing the oligonucleotides GT and NHEJ-D.

Mentions: We have shown that Polµ is able to join two DNA ends in the absence of any NHEJ accessory factors such as Ku or XRCC4/LigIV [(27); Figure 4], to which Polµ interacts via its BRCT domain (15), suggesting that Polµ could participate in the alternative end-joining pathway that takes place without the need for the classic NHEJ factors. To test if this activity is inherent to the enzyme core, we tested if the Polβ-like polymerization domain of Polµ was able to perform DNA end-bridging and trans-directed polymerization. For this, we prepared a deletion mutant lacking the BRCT domain (Polµ-ΔBRCT; see ‘Materials and Methods’ section) and confirmed, as a control, that its gap-filling activity (both efficiency and fidelity) were comparable to those of the wild-type enzyme (Figure 8A). DNAseI footprinting experiments on a DNA gap revealed a protection caused by the Polµ core (Figure 8B, lane 4) which spans a total of 10 nt of the template strand: five involved in base pairing the primer strand, one free base (the templating nucleotide in the gap; number 41) and four bases paired to the downstream strand. This protection matches that obtained with Polβ on the 5′-P-containing gap (Figure 8B, compare lanes 2 and 3). Moreover, these footprints are fully compatible with the 3D structural information available for the ternary complexes of both DNA polymerases (PDB IDs: 2IHM for Polµ and 1BPY for Polβ; Supplementary Figure S1). As already noted, by using the full-length (i.e. BRCT-containing) Polµ, the footprint is further extended (6 nt) toward the downstream strand (Figure 8B, lane 5), thus confirming that this extension is due to the intrinsic binding of the BRCT domain, downstream to the core, to a gapped DNA substrate.Figure 8.


DNA-binding determinants promoting NHEJ by human Polμ.

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

The BRCT domain of Polµ contributes, via DNA binding, to its intrinsic ability of joining DNA ends. (A) Gap-filling reactions were performed as described in ‘Materials and Methods’ section for the indicated proteins (25 nM) using a gapped substrate containing the oligonucleotides SP1C, T28 and D12. When indicated, dNTPs were added separately at 10 nM in the presence of 2.5 mM MgCl2. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and mutant and wild-type Polµ (1.5 µg). The DNA substrate used, formed by hybridization of the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) may have (P) or lack (OH) a phosphate group at the 5′-end of the downstream strand. (C) NHEJ reactions were performed as described in ‘Materials and Methods’ section, with 200 nM Polµ and using two sets of substrates: the labeled substrates were formed by hybridization of GT or C with NHEJ-D, and the cold substrates, by hybridization of either CA or C with NHEJ-D. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 10 µM in the presence of 2.5 mM MgCl2. The efficiency of the polymerization reaction is defined as a function of the percentage of primer extension. We used two different software programs to obtain the measurement of the extended primer in a minimum of three different replica experiments. (D) EMSA was performed with the indicated proteins (300 and 600 nM) using a 3′-protruding substrate containing the oligonucleotides GT and NHEJ-D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks896-F8: The BRCT domain of Polµ contributes, via DNA binding, to its intrinsic ability of joining DNA ends. (A) Gap-filling reactions were performed as described in ‘Materials and Methods’ section for the indicated proteins (25 nM) using a gapped substrate containing the oligonucleotides SP1C, T28 and D12. When indicated, dNTPs were added separately at 10 nM in the presence of 2.5 mM MgCl2. (B) Footprinting assay of the wild-type Polβ (1.5 µg) and mutant and wild-type Polµ (1.5 µg). The DNA substrate used, formed by hybridization of the oligonucleotides FP-T (template, labeled at its 5′-end), FP-P (primer) and FP-D (downstream) may have (P) or lack (OH) a phosphate group at the 5′-end of the downstream strand. (C) NHEJ reactions were performed as described in ‘Materials and Methods’ section, with 200 nM Polµ and using two sets of substrates: the labeled substrates were formed by hybridization of GT or C with NHEJ-D, and the cold substrates, by hybridization of either CA or C with NHEJ-D. The dark gray spheres indicate the presence of a 5′-P group in the downstream strand of the substrate. When indicated, dNTPs were added separately at 10 µM in the presence of 2.5 mM MgCl2. The efficiency of the polymerization reaction is defined as a function of the percentage of primer extension. We used two different software programs to obtain the measurement of the extended primer in a minimum of three different replica experiments. (D) EMSA was performed with the indicated proteins (300 and 600 nM) using a 3′-protruding substrate containing the oligonucleotides GT and NHEJ-D.
Mentions: We have shown that Polµ is able to join two DNA ends in the absence of any NHEJ accessory factors such as Ku or XRCC4/LigIV [(27); Figure 4], to which Polµ interacts via its BRCT domain (15), suggesting that Polµ could participate in the alternative end-joining pathway that takes place without the need for the classic NHEJ factors. To test if this activity is inherent to the enzyme core, we tested if the Polβ-like polymerization domain of Polµ was able to perform DNA end-bridging and trans-directed polymerization. For this, we prepared a deletion mutant lacking the BRCT domain (Polµ-ΔBRCT; see ‘Materials and Methods’ section) and confirmed, as a control, that its gap-filling activity (both efficiency and fidelity) were comparable to those of the wild-type enzyme (Figure 8A). DNAseI footprinting experiments on a DNA gap revealed a protection caused by the Polµ core (Figure 8B, lane 4) which spans a total of 10 nt of the template strand: five involved in base pairing the primer strand, one free base (the templating nucleotide in the gap; number 41) and four bases paired to the downstream strand. This protection matches that obtained with Polβ on the 5′-P-containing gap (Figure 8B, compare lanes 2 and 3). Moreover, these footprints are fully compatible with the 3D structural information available for the ternary complexes of both DNA polymerases (PDB IDs: 2IHM for Polµ and 1BPY for Polβ; Supplementary Figure S1). As already noted, by using the full-length (i.e. BRCT-containing) Polµ, the footprint is further extended (6 nt) toward the downstream strand (Figure 8B, lane 5), thus confirming that this extension is due to the intrinsic binding of the BRCT domain, downstream to the core, to a gapped DNA substrate.Figure 8.

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