Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

Abstract

DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.

Keywords: DNA damage; DNA double-strand break; DNA ligase; DNA nuclease; DNA polymerase; Genomic instability; Protein–protein interaction.

Fig. 1. Recruitment of Pol4 and Rad27 to an in vivo DSB: Effect of genetic inactivation of NEJ1 and LIF1

Kinetics of recruitment of; (A), Pol4; (B), Rad27 to the MAT DSB in the wild type SLY1A strain (diamond), SLY1A nej1Δ (square), SLY1A lif1Δ (triangle) and SLY1A nej1Δlif1Δ (circle) mutant strains was measured by chromatin immunoprecipitation as described in Experimental Procedures. Data represent the mean± S.D. of three or more independent experiments.

Fig. 2. Nej1 physically interacts with both Pol4 and RAD27; interaction of Nej1 with Pol4 is dependent upon the Pol4 BRCT domain

(A) Extracts (200 μg) from a yeast strain expressing CBP-Nej1 were incubated with; lane 4, nickel beads (Control); lane 5, nickel beads liganded by his-tagged Rad27 (1 μg, His-Rad27); lane 6, his-tagged Pol4 (1 μg, His-Pol4). Lane 1, 50 ng Rad27; lane 2, 50 ng Pol4 and lane 3, 10 μg yeast extract. (B) Extracts (200 μg) from yeast strain expressing CBP-Nej1 were incubated with; lane 4, nickel beads (Control); lane 5, nickel beads liganded by His-tagged Pol4 (1 μg, His-Pol4); lane 6, His-tagged Pol4 lacking the C-terminal BRCT domain (1 μg, His-Pol4ΔBRCT). Lane 1, 10 μg yeast extract Rad27; lane 2, 50 ng Pol4 and lane 3, 50 ng Pol4ΔBRCT. After washing, proteins were eluted with SDS sample buffer. Rad27, Pol4 and Nej1 were detected in the eluates by immunoblotting with anti-His (upper panel) and anti-CBP antibody (lower panel).

Fig. 3. The C-terminal region of Nej1 mediates the interaction with Pol4

(A) Purified proteins (50 ng of each); GST-Pol4 (lane 1, 5% of input); full-length Nej1 (lane 2); Nej1 N-terminal fragment (lane 3); Nej1 C-terminal fragment (lane 4). Purified GST-Pol4 (1 μg) was incubated with nickel beads liganded by; lane 5, no protein; lane 6, full-length Nej1 (Nej1 full length); lane 7, N-terminal fragment of Nej1 (Nej1 N terminal); lane 8, C-terminal fragment of Nej1( Nej1 C terminal) as described in Experimental Procedures. (B) Purified GST protein (lane 1, 4% of input) was incubated with nickel beads liganded by; no protein (lane 2); his-tagged Nej1 (lane 3); his-tagged Nej1 in the presence of 50 μg/ml ethidium bromide (lane 4). Purified GST-Pol4BRCT protein (lane 5, 4% of input) was incubated with nickel beads liganded by; no protein (lane 6); his-tagged Nej1 (lane 7); his-tagged Nej1 in the presence of 50 μg/ml ethidium bromide (lane 8). GST tagged proteins were detected by immunoblotting with GST antibodies. Under these conditions, Nej1 cross-reacted with the GST antibody.

Fig. 4. The C-terminal region of Nej1 mediates the interaction with Rad27

Purified proteins (50 ng of each); GST-Rad27 (lane 1, 5% of input); full-length Nej1 (lane 2); Nej1 N-terminal fragment (lane 3); Nej1 C-terminal fragment (lane 4). Purified GST-Pol4 (1 μg) was incubated with nickel beads liganded by; lane 5, no protein; lane 6, full-length Nej1 (Nej1 full length); lane 7, N-terminal fragment of Nej1 (Nej1 N terminal); lane 8, C-terminal fragment of Nej1 (Nej1 C terminal). GST- and his-tagged fusion proteins were detected by immunoblotting with GST (upper panel) and His (lower panel) antibodies.

Fig. 5. Nej1 stimulates gap-filling DNA synthesis by Pol4

(A) One of two linear DNA duplexes with partially complementary ends that anneal to form a 3-nucleotide gap was end-labeled as indicated (0.1 pmol of each). The DNA substrate (0.1 pmol, lane 1) was incubated for 1 h at 25^oC with Pol 4 (0.4 pmol, lanes 2–10) in the absence (lanes 2, 5 and 8) and presence of; lane 3, 0.4 pmol full length Nej1 (Nej1F); lane 4, 0.8 pmol full length Nej1; lane 6, 0.4 pmol Nej1 N-terminal fragment (Nej1N); lane 7, 0.8 pmol Nej1 N-terminal fragment; lane 9, 0.4 pmol Nej1 C-terminal fragment (Nej1C); lane 10, 0.8 pmol Nej1 C-terminal fragment. (B) Linear DNA duplex with an internal 1-nucleotide gap was end-labeled as indicated. The DNA substrate (1 pmol, lane1) was incubated for 1 h at 25°C with Pol4 (0.4 pmol, lanes 2–10) in the absence (lanes 2, 5 and 8) and presence of; lane 3, 0.4 pmol full length Nej1 (Nej1F); lane 4, 0.8 pmol full length Nej1; lane 6, 0.4 pmol Nej1 N-terminal fragment (Nej1N); lane 7, 0.8 pmol Nej1 N-terminal fragment; lane 9, 0.4 pmol Nej1 C-terminal fragment (Nej1C); lane 10, 0.8 pmol Nej1 C-terminal fragment. After separation by denaturing gel electrophoresis, labeled oligonucleotides in the gel were detected by PhosphorImager analysis. The boxes in Panel A indicate the grouping of images from different parts of the same gel. The positions of the substrate and extended products are indicated on the left. The results of three independent experiments are shown graphically with error bars indicating the standard deviation from the mean.

Fig. 6. Effects of human XLF and yeast Ku on gap-filling DNA synthesis by Pol4

One of two linear DNA duplexes with partially complementary ends that anneal to form a 3-nucleotide gap was end-labeled as indicated (0.1 pmol of each). (A) The DNA substrate (0.1 pmol, lane 1) was incubated for 1 h at 25°C with Pol 4 (0.3 pmol), full length yeast Nej1 (yNej1F, 1.2 pmol) and human XLF (hXLF, 1.2 pmol) as indicated. (B) Similar reactions were carried out in the absence or presence of yKu70–yKu80 (0.4 pmol) as indicated. After separation by denaturing gel electrophoresis, labeled oligonucleotides in the gel were detected by PhosphorImager analysis. The positions of the substrate and extended products are indicated on the left.

Fig. 7. Nej1 stimulates Rad27 nuclease activity

(A) One of two linear DNA duplexes with partially complementary ends that anneal to form a 5′ 3-nucleotide flap was end-labeled as indicated (0.1 pmol of each). The DNA substrate (0.1 pmol, lane 1) was incubated for 1 h at 25°C with Rad27 (0.4 pmol, lanes 2–10) in the absence (lanes 2, 5 and 8) and presence of; lane 3, 0.4 pmol full length Nej1 (Nej1F); lane 4, 0.8 pmol full length Nej1; lane 6, 0.4 pmol Nej1 N-terminal fragment (Nej1N); lane 7, 0.8 pmol Nej1 N-terminal fragment; lane 9, 0.4 pmol Nej1 C-terminal fragment (Nej1C); lane 10, 0.8 pmol Nej1 C-terminal fragment. (B) Linear DNA duplex with an internal 5′ 3-nucleotide flap was end-labeled as indicated (1 pmol). The DNA substrate (1 pmol, lane 1) was incubated for 1 h at 25°C with Rad27 (0.4 pmol, lanes 2–10) in the absence (lanes 2, 5 and 8) and presence of; lane 3, 0.4 pmol full length Nej1 (Nej1F); lane 4, 0.8 pmol full length Nej1; lane 6, 0.4 pmol Nej1 N-terminal fragment (Nej1N); lane 7, 0.8 pmol Nej1 N-terminal fragment; lane 9, 0.4 pmol Nej1 C-terminal fragment (Nej1C); lane 10, 0.8 pmol Nej1 C-terminal fragment. After separation by denaturing gel electrophoresis, labeled oligonucleotides in the gel were detected by PhosphorImager analysis. The positions of the substrate and products of nucleolytic digestion are indicated on the right. The results of three independent experiments are shown graphically with the error bars indicating the standard deviation from the mean.

Fig. 8. Effects of human XLF and yeast Ku on Rad27 nuclease activity

(A) One of two linear DNA duplexes with partially complementary ends that anneal to form a 5′ 3-nucleotide flap was end-labeled as indicated (0.1 pmol of each). The DNA substrate (0.1 pmol, lane 1) was incubated for 1 h at 25°C with Rad27 (1.2 pmol), full length yeast Nej1 (yNej1F, 2.4 pmol) and human XLF (hXLF, 2.4 pmol) as indicated. (B) Similar reactions were carried out in the absence or presence of yKu70–yKu80 (0.4 pmol) as indicated. After separation by denaturing gel electrophoresis, labeled oligonucleotides in the gel were detected by PhosphorImager analysis. The positions of the substrate, flap cleavage products and molecular mass markers of 3 and 23 nucleotides are indicated. The boxes in panel A indicate the grouping of images from different parts of the same gel.

Fig. 9. Effect of Nej1 on the processing and ligation of NHEJ intermediate with a mismatched 5′ end in reconstituted reactions with Pol4, Rad27 and Dnl4/Lif1

(A) One of two linear DNA duplexes with partially complementary ends that anneal to generate a 3-nucleotide gap and 3-nucleotide 5′ flap was end-labeled as indicated (0.1 pmol of each) to monitor removal of the mismatched 5′ flap. The labeled DNA substrate (0.1 pmol, lane 1) was incubated at 25°C for 1 hour with 0.4 pmol Rad27, 0.4 pmol Pol4 and 0.4 pmol Dnl4/Lif1 in the absence (lane 2) and presence of Nej1 (lane 3, 0.4 pmol; lane 4, 0.8 pmol) as described in Experimental Procedures. After separation by denaturing gel electrophoresis, labeled oligonucleotides in the gel were detected by PhosphorImager analysis. The positions of the 51-nt substrate and 3-nt cleavage product are indicated on the right. The results of three independent experiments are shown graphically with the error bars indicating the standard deviation from the mean. (B) To monitor gap-filling DNA synthesis and ligation, the same DNA substrate (0.1 pmol, lane 1) was end-labeled as indicated and then incubated at 25°C for 1 hour with 0.4 pmol Rad27, 0.4 pmol Pol4 and 0.4 pmol Dnl4/Lif1 in the absence (lane 2) and presence of Nej1 (lane 3, 0.4 pmol; lane 4, 0.8 pmol) as described in Experimental Procedures. After separation by denaturing gel electrophoresis, labeled oligonucleotides in the gel were detected by PhosphorImager analysis. The positions of the 50-nt substrate, 53-nt intermediate generated by gap-filling DNA synthesis and the 89-nt ligation product are indicated on the right. The results of three independent experiments are shown graphically with the error bars indicating the standard deviation from the mean.