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J. Biol. Chem., Vol. 280, Issue 32, 29030-29037, August 12, 2005

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DNA Joint Dependence of Pol X Family Polymerase Action in Nonhomologous End Joining^*

James M. Daley, Renee L. Vander Laan, Aswathi Suresh, and Thomas E. Wilson¶

From the Graduate Program in Cellular and Molecular Biology and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602

Received for publication, May 13, 2005 , and in revised form, June 14, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Double strand breaks (DSBs)¹ can be repaired by two mechanisms: homologous recombination and nonhomologous end joining (NHEJ) (reviewed in Ref. 1). Although recombination utilizes an intact homologous template to promote accurate repair, NHEJ relies on base-pairing potential in the terminal overhangs, termed microhomologies, to guide religation of the ends. NHEJ is initiated when the Ku heterodimer (Yku70/Yku80 in yeast) binds to the DSB ends. The Mre11-Rad50-Xrs2 complex is thought to tether the two termini together, along with DNA-PK in mammalian cells (2–4). The DNA ligase IV complex in yeast, consisting of the catalytic subunit Dnl4 and its cofactor Lif1, ligates the break to restore the duplex (5).

Agents that create DSBs often leave damaged bases at the break termini (6, 7). Processing enzymes, at minimum including a nuclease and a polymerase, must remove and resynthesize these damaged bases before ligation can occur, but such repair is ultimately accurate. The nuclease Artemis is involved in mammalian NHEJ (8), but in yeast the NHEJ nuclease(s) are unknown. The Pol X family of DNA polymerases has been implicated in NHEJ. Pol4, the only Pol X family member in yeast, is required for gap filling in some end configurations but not others (9). Mammals have four Pol X family members: Pol, Pol, Polµ, and terminal deoxynucleotidyl transferase (reviewed in Ref. 10). Pol functions in base excision repair (11), whereas terminal deoxynucleotidyl transferase is specifically associated with V(D)J recombination, a specialized form of NHEJ that occurs only in B- and T-cell maturation (12, 13). In contrast, recent reports have implicated Pol and Polµ in general NHEJ. Immunodepletion of Pol but not Polµ inhibits in vitro NHEJ (14). Additionally, Polµ has been show to interact with Ku and the XRCC4-ligase IV complex and is recruited to DNA damage foci (15). Both Pol and Polµ can act in NHEJ in a reconstituted system (16). In all cases, Pol X polymerases are only required for NHEJ when gaps must be filled, indicating that they are not part of the core NHEJ complex.

Although the ability of NHEJ to trim and fill overhangs is of great importance to restorative repair, it also enables imprecise rejoining. NHEJ faithfully repairs DSBs with compatible overhangs the majority of the time, but Pol4-dependent inaccurate repair involving overhang mispairing and gain or loss of nucleotides can occur (17–19). NHEJ can also join incompatible termini by utilizing microhomologies either in the DSB overhangs or exposed by resection into the duplex DNA (9, 20). Because incompatible termini necessarily originate from separate or resected DSBs, such imprecise NHEJ events lead to chromosomal rearrangements or deletions. Together, terminal base damage, overhang mispairing, and incompatible termini lead to a large variety of processed NHEJ joints and outcomes in vivo. Thus, understanding the mechanistic basis of processed NHEJ, including the differential contributions of processing enzymes to various joint types, is of great importance.

We previously identified some Pol4-dependent NHEJ events but, because of limited ability to create specific DSB termini, could not identify the precise characteristics of a joint structure that necessitated Pol4 action (9). Here, we sought to determine why only some joints that require gap filling are Pol4-dependent by using oligonucleotide-modified plasmids (OMPs) to perform a systematic analysis of DSB end configurations (21). We found that only NHEJ events that involve 3'-overhangs and require gap filling on both strands are strongly Pol4-dependent, with Pol4 promoting accurate repair. Repair of Pol4-independent gap-containing DSBs does not require the translesion polymerases Pol or Pol, implicating the replicative polymerases in these joints. Pol4 appears to be exclusively associated with the NHEJ pathway of DSB repair, because it is not required for Ku-independent DSB rejoining that occurs via long overhangs or recessed microhomologies (i.e. microhomologies within the duplex portion of a DSB). Finally, we extended these findings to mammalian NHEJ by showing that Pol, Pol, and Polµ can each partially replace Pol4 when expressed in yeast. Interestingly, these mammalian polymerases reconstituted NHEJ to different extents depending on the joint structure. These data suggest that specialized NHEJ polymerases are recruited to DSBs with termini that are too unstable to utilize the replicative polymerases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Yeast Strains—Saccharomyces cerevisiae strains used for plasmid transformation were isogenic derivatives of the previously described wild-type strain YW389 (MAT ade2D0 his3D200 leu2 lys2–801 trp1D63 ura3D0) (22). YW438 (pol4::MET15), YW459 (yku70::HIS3), YW507 (dnl4::MET15), YW1567 (pol4::MET15 rad30::kanMX4), YW1568 (pol4::MET15 rev3::HIS3), YW1569 (pol4::MET15 rad30::kanMX4 rev3::HIS3), and YW1654 (pol4::MET15 yku70::kanMX4) were created using a PCR-mediated one-step gene replacement technique (23). All disruptions were confirmed by PCR. The suicide deletion strains YW1276 (ADE2::HOSD[+1]) and YW1407 (ADE2::HOSD[+1] pol4::URA3) were described previously (18). When indicated, yeast were transformed with the appropriate polymerase expression plasmids (see below) with continued selection for the plasmid using growth medium lacking leucine.

Polymerase Expression Plasmids—Polymerase expression plasmids were created by gap repair with tailed PCR fragments into the CEN/LEU2 plasmid pTW435, which uses the ADH1 promoter to drive expression of a nuclear localization signal (NLS)-Myc-tagged protein. The POL4 coding sequence was amplified from yeast genomic DNA, and the Pol, Pol, and Polµ sequences were amplified from human placental cDNA (Clontech) using the Advantage HF PCR kit (BD Biosciences). Plasmids were sequenced to verify the correct coding sequence.

Oligonucleotide-modified Plasmids—OMPs were created as described previously (21). Briefly, two pairs of annealed oligonucleotides designed to restore the ADE2 coding sequence in plasmid pTW423 were ligated onto the ends of pTW423 digested with BglII and XhoI (supplemental Fig. 1A). Plasmids were purified from free oligonucleotides by agarose gel electrophoresis, and product size, linearity, and integrity were verified. DNA concentration was quantified by UV spectrometry. Because the DSB side of the oligonucleotides initially bore 5'-hydroxyls to prevent ligation of the DSB in vitro, purified OMPs were typically treated with T4 polynucleotide kinase (New England Biolabs) followed by a second round of purification. The extent of oligonucleotide ligation was then monitored by primer extension (supplemental Fig. 1B). Because the ligation extent is not constant, small differences in NHEJ efficiency between OMPs cannot be considered significant, although large differences likely reflect real changes in the repair rate. Interstrain differences with a given OMP are independent of this effect.

Preparation and SfiI Digest of Plasmids pTW502–507—Plasmids pT-W502–507 are derivatives of the CEN plasmid pES16, which was described previously (5). Two PCR products amplified from pES16 were prepared to insert an SphI site and stop codons in all reading frames flanked by two SfiI sites after the second codon of ADE2. The first product was made using primers OW2003 (5'-ACACCCGCCGCGCTTAATG-3') and OW2041 (5'-TACTAGCATGCGGCCGAGTGGGCCATCCATACTTGATTGTTTTGT-3'). The second product was made with OW2068–2073, depending on the plasmid (5'-TACTAGCATGCXTAACTGACTAGGCCCXXXCGGCCCATCTAGAACAGTTGGTATATTAGGA-3') and OW2008 (5'-TTAACAGATCTCACAATCATGACTGC-3'). The SphI site is indicated in italics, the SfiI sites are in bold, stop codons are underlined, and variable nucleotides that place ADE2 in-frame or make up the desired overhang are indicated by X. The products were digested with NotI and SphI or BglII and SphI, respectively, and simultaneously ligated into NotI- and BglII-digested pES16. Plasmids were then digested with SfiI to excise the polyterminator and gel-purified as above.

Yeast Transformation—Plasmids were transformed into yeast using a high efficiency lithium acetate method as described previously (21). 100 ng of linear plasmid (marked with URA3) was co-transformed with 10 ng of supercoiled pRS315 as a transformation control (marked with LEU2). Cells were plated in parallel to glucose medium lacking either uracil or leucine and grown at 30 °C for 3 days. The relative repair efficiency for a strain-plasmid combination is expressed as the ratio of Ade+ (white) colonies on plates lacking uracil, which represent inframe joining of DSBs, to colonies on plates lacking leucine. Note that although in-frame joining effectively selected for the target joint in most cases, alternative Ade+ joints can also occur and will be represented in the graphs. To ensure adequate colony counts, more cells were plated for less efficient joints and/or mutant strains. A minimum of 150, and more typically 500–1200, Ade+ colonies were counted for each joint in wild-type yeast, except where noted. Similar colony numbers were counted for mutant strains when possible, but in instances in which the mutant strain was highly deficient at forming a particular joint, counts were made of the few colonies recovered at the highest plating density. Individual data panels always represent results collected in parallel with a single preparation of the plasmids and carrier DNA to allow valid comparisons. Small variances between graphs are the result of different reagent sets and are not significant as compared with the large changes observed when comparing mutant and wild-type strains within a graph.

Joint Analysis—To determine whether Ade+ colonies had formed the target joint in wild-type and/or residual colonies of mutant strains, a sampling of independent colonies for relevant plasmid-strain combinations was subjected to joint analysis. For this, PCR was used to amplify the region of ADE2 containing the break followed by sequencing. A complete understanding of a particular joint efficiency can be determined only by considering both the transformation efficiency (Ade+/Leu+ ratio) and pattern of joints among sequenced colonies.

Suicide Deletion—The HO(+1) suicide deletion allele was used to measure the percentage of cells that undergo NHEJ and the percentage of imprecise +2 NHEJ events as described previously (18).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Pol4 Is Required for Gap Filling Independently of Nonhomologous Flaps—On the basis of previous data (9), we considered several joint features, in addition to the presence of gaps, as possible explanations for Pol4 dependence in NHEJ: formation of nonhomologous flaps (i.e. unpaired terminal nucleotides) upon overhang annealing, the stability of the annealed overhangs, and overhang polarity. To test whether the presence of nonhomologous flaps causes Pol4 to be required for gap filling, a series of plasmids were created that bore the four possible combinations of 1-nt flaps and gaps (Fig. 1). The fully compatible and gap-only DSBs are the most physiologically relevant of these as they correlate with precise repair of complementary overhangs at a single DSB, at which gaps would result from the removal of damaged terminal nucleotides. More generally, this panel mimics end structures that might arise when incompatible termini of different DSBs are joined. To make these substrates we took advantage of the fact that the 3-base SfiI 3'-overhangs can be composed of any combination of nucleotides. SfiI sites were placed after the start codon of ADE2 in such a way that the target joint placed ADE2 in-frame. SfiI-digested plasmids were transformed into wild-type, pol4, yku70, and dnl4 yeast to monitor repair, along with a supercoiled LEU2-marked plasmid to control for transformation efficiency. As expected, DSBs with fully compatible overhangs did not require Pol4 for joining (Fig. 1, first target joint). When a 1-nt gap was introduced, joining became strongly Pol4-dependent (Fig. 1, second target joint). This joint was also highly dependent on Dnl4 and Yku70, indicating that the repair occurred by NHEJ. The few Ade+ colonies that did arise in pol4 yeast utilized other short recessed microhomologies. A joint with a 1-nt gap and a 1-nt nonhomologous flap also showed substantial Pol4 dependence, with the target overhang-to-overhang joint not recovered among five sequenced residual events from the pol4 strain (Fig. 1, fourth target joint). Thus, Pol4 is required for gap filling in 3'-overhang NHEJ events regardless of the presence or absence of nonhomologous flaps. A joint with 1-nt flaps but no gap showed only a 2-fold reduction in the pol4 strain (Fig. 1, third target joint), similar to previous results (9).

View larger version (41K): [in this window] [in a new window]   FIG. 1. Pol4 is required for gap filling during NHEJ of 3'-overhangs regardless of whether nonhomologous flaps are present. Plasmids cut with SfiI were transformed into yeast. The Ade+ outcome selected for NHEJ events predicted to occur in the indicated joint configurations (as well as other joints in the same relative reading frame). Repair frequencies for each genotype are expressed as the ratio of Ade+ colonies over Leu+ colonies resulting from the uptake of a cotransformed supercoiled plasmid (top panel). Each bar represents the mean ± S.D. from at least three independent transformations performed with the same reagents. Samples of independent joints were recovered, amplified, and sequenced with primers within ADE2 (bottom 4 panels). Bases annealed in the joint are shown in bold, vertical lines indicate the location of the overhangs, and dashes indicate gaps in the sequence. Light face letters highlight bases that diverged from the expected plasmid sequence. Numbers (e.g. "TGG" right of center in third target joint) 5x) indicate how many times a joint was recovered for a given genotype. Assessment of the efficiency of any particular joint must consider both the transformation efficiency and its frequency in the recovered sequences.

Pol4 Is Required at All NHEJ-dependent Microhomology Lengths—We next asked whether the pattern of Pol4 dependence seen in Fig. 1 would be recapitulated if the joint contained a more stable (but still NHEJ-dependent) microhomology. The OMP approach was used to create DSBs with the desired overhang configurations. In this system, annealed oligonucleotides are ligated onto restriction site ends within the ADE2 gene of the plasmid pTW423 (supplemental Fig. 1A). Ade positivity largely but not completely selects for the target joint by requiring ligation of both oligonucleotide pairs and subsequent rejoining of the DSB (21). We again created a panel of DSBs containing 3'-overhangs and each possible combination of 1-base gaps and 1-base flaps but now in the context of a 4-base microhomology. The overhang sequence was kept constant to ensure that differences in repair were due to the presence of flaps and gaps. Such ends are again within the spectrum expected to occur at naturally occurring DSBs, with flaps relevant only to mutagenic events. These DSBs showed a pattern of Pol4 dependence similar to the shorter overhangs. Each gap-containing joint was again highly Pol4-dependent regardless of whether flaps were present (Fig. 2A). The deficiency in the pol4 strain was comparable with that of the dnl4 strain, indicating that Pol4 is strongly required for NHEJ of gap-containing DSBs with 3'-overhangs. Residual Ade+ colonies generated by the pol4 strain corresponded to the designed joint, however, indicating that this event can occur in the absence of Pol4 at a low frequency (Fig. 2A). A joint with flaps but no gaps again showed slightly decreased joining in the absence of Pol4 (Fig. 2A, third target joint). Thus, Pol4 is required for efficient gap filling at 3'-overhangs regardless of whether the overhang contains 1, 2, or 4 homologous bases and in all cases tends to promote accurate rejoining of the microhomology present in the overhangs.

Overhang Polarity Determines Pol4 Dependence—We next generated a comparable panel of OMPs with 5'-overhangs to test the importance of overhang polarity, given that each polarity is expected to occur naturally. These DSBs were markedly less Pol4-dependent than the corresponding DSBs with 3'-overhangs (Fig. 2B). When the DSB contained 1-nt gaps, a 2.1-fold decrease in joining efficiency was observed in the pol4 strain as compared with a 39-fold deficiency with 3'-overhangs. Similarly, a 5'-overhang joint with 1-nt flaps and 1-nt gaps was reduced by only 1.6-fold in a pol4 mutant versus 31-fold with 3'-overhangs. Sequencing revealed that Ade+ colonies indeed represented the designed joint in both wild-type and pol4 strains for 5'-overhangs (Fig. 2B). Dnl4 was still required for 5'-overhang joining, however, indicating that repair still occurred by bona fide NHEJ (Fig. 2B). Thus, the NHEJ requirement for Pol4 is greatly relaxed at 5'-overhangs.

Why might 3'-overhang joints depend more strongly on a specialized NHEJ polymerase? The 3'-primer terminus that must be extended during gap filling has a different stability and orientation when comparing 3'- and 5'-overhangs. With 5'-overhangs, the 3'-end is on the stable duplex adjacent to the gap, and synthesis proceeds centrally. With 3'-overhangs, the polymerase must synthesize away from the break center using the overhang-to-overhang base pairing as the primer-template pair. This duplex consists of only 1 to at most 6 bases (21), and is therefore much less stable than the primer used for 5'-overhang filling. Thus, Pol4 is likely a specialized polymerase that evolved to function in circumstances in which an unstable duplex is the only available primer for DNA synthesis. The recently solved crystal structure of Pol, the mammalian Pol X polymerase most related to Pol4, supports this idea (25). Interactions between the polymerase and the 3'-primer terminus are much less extensive in Pol than in the replicative polymerases, whereas the lyase domain of Pol makes extensive contacts with the base on the 5'-side of the gap. The unique biochemical properties of Pol X polymerases, including terminal deoxynucleotidyl transferase activity and frequent primer-template slippage, appear to reflect this reduced dependence on a stably annealed primer (26–28). It is also possible that accessibility of the primer terminus within the bound NHEJ complex differs with joint polarity and that only Pol4 is capable of gaining access to internal 3'-ends via interactions with other NHEJ proteins. Indeed, 5'-overhangs could even be filled prior to association of the DSB ends, whereas 3'-overhangs can only be filled during NHEJ.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Overhang polarity determines Pol4 dependence. A, OMPs with 3'-overhangs for which the Ade+ phenotype selects for the indicated combinations of flaps and gaps. Repair frequencies (top panel) and recovered joints (bottom panel) are shown as in Fig. 1. B, rejoining of similar DSBs with 5'-overhangs. C, the translesion polymerases are not required for rejoining 5'-overhang DSBs containing gaps. Note that different carrier DNA preparations were used in B and C, resulting in a slightly different range in the transformation ratio because of non-equivalent effects of carrier DNA on the uptake of linear and supercoiled DNA.

View larger version (31K): [in this window] [in a new window]   FIG. 3. DSBs containing one ligatable strand and one gapped strand are repaired in the absence of Pol4. DSBs with mixed nicks and gaps were generated using OMPs that selected for the indicated joints, with data shown as in Fig. 1.

Pol4 Is Dispensable When Only One Strand Requires Gap Filling—To gain insight into the order of events in NHEJ, we next created DSBs identical to the gap-containing 3'-overhang used in Fig. 2A except with a gap on only one strand. In this configuration, Pol4 was largely dispensable for joining, but Dnl4 was not (Fig. 3). Joint sequences showed that the duplex was restored as expected in both wild-type and pol4 strains (Fig. 3). This indicates that even in the absence of Pol4, the NHEJ machinery can ligate the one nicked strand, as has been observed in vitro (16). It is possible that the now stable primer could be extended by another polymerase to fill the gap during NHEJ, but it seems more likely that disengagement of the NHEJ proteins results in a single-stranded 1-nt gap. This would not affect the outcome because the plasmid could be replicated by the copying of the intact strand or could simply be repaired by base excision repair because the critical need for reassociation of DSB ends has been satisfied.

View larger version (30K): [in this window] [in a new window]   FIG. 4. NHEJ that utilizes a recessed microhomology does not require Pol4. DSBs with or without a recessed microhomology were generated using OMPs that selected for the indicated joints, with data shown as in Fig. 1.

Pol4 Can Fill Gaps Greater than 1-nt in Length—Pol X polymerases are able to efficiently fill gaps of various lengths in vitro as long as a 5'-terminus is provided (27, 35). To test this ability during NHEJ, OMPs similar to those used in Fig. 2A were generated in which the 4-base microhomology was kept constant, but the gap on each side was extended to 2, 3, or 4 bases. Such DSB structures may be formed directly by DNA-damaging agents and are also thought to occur in vivo during the early stages of 5'-resection. Ends that formed 1- and 2-base gaps were joined at similar rates in wild-type yeast and showed >100-fold reductions in the pol4 and dnl4 mutant strains (Fig. 5). Extending the gap to 3 and 4 bases reduced the joining efficiency substantially in the wild-type strain (Fig. 5), consistent with the notion that resection inhibits NHEJ and commits DSB repair to recombination (36). The reduced repair rate made it difficult to determine reliably whether these events were Pol4-dependent. We nonetheless did observe more Ade+ colonies in wild-type yeast (93 for the 3-base gap and 41 for the 4-base gap) than in the pol4 strain (0 for the 3-base gap and 4 for the 4-base gap) for these DSBs. Thus, although there is an upper limit to the gap length that can be efficiently repaired by NHEJ, the few successful NHEJ events at longer gaps in wild-type yeast likely utilize Pol4.

View larger version (10K): [in this window] [in a new window]   FIG. 5. Pol4 can fill gaps greater than 1 nt during NHEJ. DSBs with 4-base microhomologies in 3'-overhangs and variable gaps on each strand were generated using OMPs that selected for the indicated joints, with data shown as in Fig. 1, top panel.

Pol4 Is Required at DSBs Containing 5'-Hydroxyls Only When Gaps Are Also Present—DSBs caused by DNA-damaging agents frequently contain 5'-hydroxyls that must be repaired before ligation can occur (6, 7). Pol4 can efficiently fill gaps with 5'-hydroxyl termini in vitro, even though Pol and Pol require a 5'-phosphate for full activity (27, 37). We therefore asked whether Pol4 is required for NHEJ of a DSB containing 3'-overhangs, a 1-nt gap, and 5'-hydroxyls. This joint was formed less efficiently than the corresponding 5'-phosphate joint, consistent with previous results (21), but remained strongly Pol4-dependent (Fig. 7, third and fourth target joints). Thus, Pol4 is indeed utilized in vivo when the NHEJ machinery encounters gaps with 5'-hydroxyl termini. But how is the 5'-hydroxyl resolved? The terminal base containing the 5'-hydroxyl could be removed by a nuclease either before or after its resynthesis by a polymerase. Initial removal of the base would result in a gapped joint, which is demonstrated above to require Pol4. Moreover, Pol4 interacts with the 5'-nuclease Rad27 in a manner consistent with the concerted repair of damaged 5' termini (38). Therefore, we next asked whether NHEJ of DSBs with 5'-hydroxyl termini but no gaps requires Pol4. Surprisingly, wild-type levels of repair were observed in the absence of Pol4 regardless of whether the termini contained 5'-hydroxyls or 5'-phosphates (Fig. 7, first and second target joints). The simplest explanation for these data is that the 5'-hydroxyl is converted to a phosphate by a kinase. Indeed, mammalian polynucleotide kinase/3'-phosphatase is thought to act during NHEJ (39, 40). Its yeast homolog, Tpp1, contains only the 3'-phosphatase domain, however, and we correspondingly failed to detect a 5'-kinase activity in yeast (22). It is thus unclear whether a cryptic kinase activity is present or the removed 5' terminal base is unexpectedly resynthesized by a polymerase other than Pol4.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Pol4 is required for gap filling only in Dnl4-dependent joints. DSBs with variable length 3'-overhangs were generated using OMPs that selected for the indicated joints, with data shown as in Fig. 1, top panel. Data are separated onto two graphs because DSBs with 8- and 10-base overhangs are repaired about 10-fold more efficiently than DSBs with shorter overhangs. The Ade+/Leu+ ratio can exceed 1 for such plasmids because 10-fold more linear plasmid than circular plasmid is transformed, and yeast take up linear and supercoiled plasmid DNA with different efficiencies.

View larger version (29K): [in this window] [in a new window]   FIG. 7. The presence of terminal 5'-hydroxyls does not affect the requirement for Pol4. DSBs with variable 5'-phosphorylation status were generated using OMPs that selected for the indicated joints, with data shown as in Fig. 1.

NHEJ was measured using the above plasmid assays as well as a previously described chromosomal assay. Broadly, the results indicated that the mammalian Pol X polymerases can indeed act during yeast NHEJ, although generally at lower efficiency. Strikingly, each polymerase showed a distinct pattern of joint formation. In the plasmid assays, a joint containing 2 homologous bases and 1-nt gaps was completed by Pol and Polµ but not by Pol (Fig. 8B, first target joint). Only Polµ was able to catalyze formation of a joint with 1 homologous base, a 1-base gap, and a 1-base flap, however (Fig. 8B, second target joint). Strains expressing Pol or Pol formed only the MMEJ event also detected by this plasmid. All of the mammalian polymerases, including Pol, were able to complete a joint containing 4 complementary bases and 1-nt gaps, although Polµ was now paradoxically less efficient (Fig. 8B, third target joint). Adding 1-nt nonhomologous flaps to this joint produced a similar pattern (Fig. 8B, fourth target joint). The chromosomal "suicide deletion" assay selects for imprecise NHEJ in the +2 reading frame at an HO endonuclease-created DSB (18). Such joining occurs in 1–2% of all surviving cells and is strictly Pol4-dependent (9, 18). When Pol4 is present, nearly all events correspond to a joint with a 2-base insertion due to mispairing of the 3'-overhangs, consistent with the above results (Fig. 8C). The mammalian Pol X polymerases each resulted in a slight but detectable increase in colony recovery compared with the pol4 strain, but these nearly all corresponded to other imprecise joints (Fig. 8C). Most common was a loss of 1 base resulting from a different overhang mispairing.

These data establish that each mammalian Pol X polymerase is capable of participating in NHEJ when expressed in yeast. The significance of this finding is most clear for Pol and Polµ. These polymerases have been implicated directly in NHEJ based on in vitro results (14, 16), and were only modestly overexpressed in yeast (Fig. 8A). Their action in NHEJ likely reflects in part their similar apparently reduced dependence on a stable primer-template pair (26, 41). Pol4, Polµ, and Pol are not biochemically identical, however (27, 28, 35), which likely explains their differential ability to catalyze various NHEJ joints. Our data are not sufficient to fully characterize the variable joining patterns, but they nonetheless support the idea that the seemingly redundant mammalian NHEJ polymerases evolved to deal with different end structures (10). Pol4, Pol, and Polµ also each contain an amino-terminal BRCA1 carboxyl-terminal domain that interacts with various components of the NHEJ core machinery (15, 42, 43). It is likely that this domain contributes to the ability of Pol and Polµ to function in yeast NHEJ, with species divergence in protein-protein interactions accounting for their lesser overall activity. We are currently exploring these possibilities. It is much less certain whether Pol is a bona fide NHEJ polymerase as it was less consistently able to complement pol4 mutation despite being expressed at a much higher level. We argue that this overexpression most likely helped Pol overcome its lack of a BRCA1 carobxyl-terminal domain. Moreover, Pol has never been functionally linked to mammalian NHEJ.

View larger version (63K): [in this window] [in a new window]   FIG. 8. Complementation of pol4 mutation by mammalian Pol X polymerases. A, expression was verified by Western blot of whole cell lysates using the 9E10 Myc antibody (Santa Cruz Biotechnology). The blot was substantially overexposed so that the low level of Pol4 could be visualized. Note the much higher expression of Pol. B, DSB rejoining was assessed by plasmid recircularization. The first two DSBs were made by SfiI digestion as in Fig. 1, and the second two were generated using OMPs as in Fig. 2. C, The HO(+2) suicide deletion assay was used to quantify the percentage of cells that undergo imprecise NHEJ of a chromosomal DSB. Repair is expressed as the fraction of all NHEJ events in the +2 reading frame (top panel). Recovered joints (bottom panel) are shown as in Fig. 1.

	FOOTNOTES

 
^* This work was supported by the Pew Scholars Program in the Biomedical Sciences of the Pew Charitable Trusts and Public Health Service Grant CA90911 (to T. E. W.) and by a University of Michigan Rackham predoctoral fellowship (to J. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

^¶ To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, Medical Science I M4214/0602, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. Tel.: 734-936-1887; Fax: 734-763-6476; E-mail: wilsonte{at}umich.edu.

¹ The abbreviations used are: DSB, double strand break; NHEJ, nonhomologous end joining; MMEJ, microhomology-mediated end joining; NLS, nuclear localization signal; nt, nucleotide; OMP, oligonucleotide-modified plasmid.

	ACKNOWLEDGMENTS

 
We thank the members of the Wilson laboratory for support, ideas, and helpful comments on the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 

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