Abasic sites linked to dUTP incorporation in DNA are a major cause of spontaneous mutations in absence of base excision repair and Rad17-Mec3-Ddc1 (9-1-1) DNA damage checkpoint clamp in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, inactivation of base excision repair (BER) AP endonucleases (Apn1p and Apn2p) results in constitutive phosphorylation of Rad53p and delay in cell cycle progression at the G2/M transition. These data led us to investigate genetic interactions between Apn1p, Apn2p and DNA damage checkpoint proteins. The results show that mec1 sml1, rad53 sml1 and rad9 is synthetic lethal with apn1 apn2. In contrast, apn1 apn2 rad17, apn1 apn2 ddc1 and apn1 apn2 rad24 triple mutants are viable, although they exhibit a strong Can(R) spontaneous mutator phenotype. In these strains, high Can(R) mutation rate is dependent upon functional uracil DNA N-glycosylase (Ung1p) and mutation spectra are dominated by AT to CG events. The results point to a role for Rad17-Mec3-Ddc1 (9-1-1) checkpoint clamp in the prevention of mutations caused by abasic (AP) sites linked to incorporation of dUTP into DNA followed by the excision of uracil by Ung1p. The antimutator role of the (9-1-1) clamp can either rely on its essential function in the induction of the DNA damage checkpoint or to another function that specifically impacts DNA repair and/or mutagenesis at AP sites. Here, we show that the abrogation of the DNA damage checkpoint is not sufficient to enhance spontaneous mutagenesis in the apn1 apn2 rad9 sml1 quadruple mutant. Spontaneous mutagenesis was also explored in strains deficient in the two major DNA N-glycosylases/AP-lyases (Ntg1p and Ntg2p). Indeed, apn1 apn2 ntg1 ntg2 exhibits a strong Ung1p-dependent Can(R) mutator phenotype with a spectrum enriched in AT to CG, like apn1 apn2 rad17. However, genetic analysis reveals that ntg1 ntg2 and rad17 are not epistatic for spontaneous mutagenesis in apn1 apn2. We conclude that under normal growth conditions, dUTP incorporation into DNA is a major source of AP sites that cause high genetic instability in the absence of BER factors (Apn1p, Apn2p, Ntg1p and Ntg2p) and Rad17-Mec3-Ddc1 (9-1-1) checkpoint clamp in yeast.     

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G2 checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-D (null) mutant is defective in both S and G2 checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G2 checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1, and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G2 checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G2-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G2 arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.     

Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae

A broad spectrum of genetic damage results from exposure to hexavalent chromium. These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions. Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood. Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality. Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment. The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression. Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested. A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay. In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality. RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment. These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest. Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment.     

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations

Summary Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (ral ⁰). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m², the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.     

HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.     

Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae

The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss.     

Nucleotide excision repair defect influences lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent in the absence of base excision repair

Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Me-lex, [1-methyl-4-[1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propane, a compound that selectively generates 3-methyladenine (3-MeA). We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER) to generate an abasic site, is significantly more sensitive to the toxicity of Me-lex with respect to wild type but shows only a marginal increase in mutagenicity. A strain defective in AP endonuclease activity (Deltaapn1apn2), also required for functional BER, is equally sensitive to the toxicity as the Deltamag1 mutant but showed a significantly higher mutation frequency. In the present work, we have explored the role of nucleotide excision repair (NER) in Me-lex-induced toxicity and mutagenicity since it is known that NER acts on abasic sites in vivo in yeast and in vitro assays. To accomplish this, we have deleted one of the genes essential for NER in yeast, namely, RAD14, both in the context of an otherwise DNA repair-proficient strain (Deltarad14) and in a BER-defective isogenic derivative lacking the MAG1 gene (Deltamag1rad14). Interestingly, no sensitivity to the treatment with Me-lex was conferred by the simple deletion of RAD14. However, a significant enhancement in toxicity and mutagenicity was observed when cells lacked both Rad14 and Mag1. The mutation spectrum induced by Me-lex in the Deltamag1rad14 strain is indistinguishable from that observed in the Deltaapn1/Deltaapn2 or in the Deltamag1 strains. The results indicate that in yeast NER can play a protective role against 3-MeA-mediated toxicity and mutagenicity; however, the role of NER is appreciable only in a BER-defective background.     

Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae.

The bifunctional alkylating anticancer drug nitrogen mustard forms a variety of DNA lesions, including monoadducts and intrastrand and interstrand crosslinks. Although it is known that nucleotide excision repair (NER) is important in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined. Using isogenic Saccharomyces cerevisiae strains disrupted for a variety of NER and BER genes we have examined the relative importance of the two pathways in the repair of nitrogen mustard adducts. As expected, NER defective cells (rad4 and rad14 strains) are extremely sensitive to the drug. One of the BER mutants, a 3-methyladenine glycosylase defective (mag1) strain also shows significant hypersensitivity. Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity. Furthermore, both rad14 and mag1 disruptants show elevated levels of nitrogen mustard-induced forward mutation. Measurements of repair rates of nitrogen mustard N-alkylpurine adducts in the highly transcribed RPB2 gene demonstrate defects in the processing of mono-adducts in rad4, rad14 and mag1 strains. However, there are differences in the kinetics of adduct removal in the NER mutants compared to the mag1 strain. In the mag1 strain significant repair occurs within 1 h with evidence of enhanced repair on the transcribed strand. Adducts however accumulate at later times in this strain. In contrast, in the NER mutants repair is only evident at times greater than 1 h. In a mag1/rad4 double mutant damage accumulates with no evidence of repair. Comparison of the rates of repair in this gene with those in a different genomic region indicate that the contributions of NER and BER to the repair of nitrogen mustard adducts may not be the same genome wide.     

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.     

Abrogation of the Chk1-Pds1 checkpoint leads to tolerance of persistent single-strand breaks in Saccharomyces cerevisiae

In budding yeast, Apn1, Apn2, Tpp1, and Rad1/Rad10 are important enzymes in the removal of spontaneous DNA lesions. apn1 apn2 rad1 yeast are inviable due to accumulation of abasic sites and strand breaks with 3' blocking lesions. We found that tpp1 apn1 rad1 yeast exhibited slow growth but frequently gave rise to spontaneous slow growth suppressors that segregated as single-gene mutations. Using a candidate gene approach, we identified several tpp1 apn1 rad1 suppressors. Deleting uracil glycosylase suppressed both tpp1 apn1 rad1 and apn1 apn2 rad1 growth defects by reducing the abasic site burden. Mutants affecting the Chk1-Pds1 metaphase-anaphase checkpoint only suppressed tpp1 apn1 rad1 slow growth. In contrast, most S-phase checkpoint mutants were synthetically lethal in a tpp1 apn1 rad1 background. Epistasis analyses showed an additive effect between chk1 and ung1, indicating different mechanisms of suppression. Loss of Chk1 partially restored cell-growth parameters in tpp1 apn1 rad1 yeast, but at the same time exacerbated chromosome instability. We propose a model in which recombinational repair during S phase coupled with failure of the metaphase-anaphase checkpoint allows for tolerance of persistent single-strand breaks at the expense of genome stability.     

Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA.

Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.     

Origin of endogenous DNA abasic sites in Saccharomyces cerevisiae

Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3'-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality. Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G(2)/M checkpoint. These results point to uracil as a critical source of the formation of endogenous AP sites in DNA. Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication. Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant. Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.     

The repair of large DNA adducts in mammalian cells.

This paper describes experiments involving the measurement of DNA damage and repair after treatment with 4-nitroquinoline 1-oxide (4NQO) or aflatoxin B1 (AFB1) epoxide in a number of mammalian cell cultures primarily associated with defects in the excision repair of UV-induced DNA damage. The results with transformed derivatives of XP cells belonging to different complementation groups showed that the extent of repair of 4NQO adducts at the N2 or C8 of guanosine did not correlate to the extent of repair reported by others after UV-irradiation. An examination of 4NQO repair in rodent UV-sensitive cell lines from different ERCC groups indicated that again there was little correlation between the extent of 4NQO and UV repair. However, regardless of complementation group those mutants that were defective in the repair of pyrimidine dimers and 6,4-photoproducts did exhibit a reduced ability to repair the 4NQO N2 guanosine adduct, whereas those mutants defective in pyrimidine dimer repair alone were able to repair this lesion as normal. In all of these cell lines there was a normal capacity to repair the 4NQO C8 guanosine adduct. Less extensive experiments involving AFB1 epoxide showed an XPC-transformed cell line was able to repair 40% of lesions after 6 h, whereas only 20% of repair is seen after UV. The rodent mutant V-C4 which belongs to the same ionising radiation group as irs2, was partially defective in repairing AFB1-induced damage. These experiments highlight the fact that although there are many commonalities between the repair of UV damages and lesions classed as large DNA adducts differences clearly exist, the most striking example here being the repair of the C8 guanosine 4NQO adduct which rarely correlates with a defect in UV repair.     

Genetic analysis of ionizing radiation-induced mutagenesis in Saccharomyces cerevisiae reveals TransLesion Synthesis (TLS) independent of PCNA K164 SUMOylation and ubiquitination

Ionizing radiation-induced mutagenesis (IR-IM) underlies a basis for radiation associated carcinogenesis as well as resistance to radiation therapy. This process was examined in Saccharomyces cerevisiae using an array of isogenic DNA repair deficient mutants. Mutations inactivating homologous recombination (rad51, 52, 54) or nucleotide excision repair (rad1, rad10, rad4) caused elevated IR-IM whereas inactivation of TransLesion Synthesis (TLS: rad6) caused severely defective IR-IM. Of the mutations inactivating TLS polymerases, rev3 and rev1 caused equally severe defects in IR-IM whereas rad30 did not significantly affect the process. The effects of the rev3, rev1, and rad6 mutations on IR-IM were epistatic, suggesting the requirement of both polymerase zeta and Rev1p in IR-IM related TLS. Although PCNA K164 SUMOylation/ubiquitination is a proposed prerequisite for TLS, the IR-IM defect of a rev3 or a rad6 mutant was worse than and epistatic to the pol30K164R mutant, a mutant in which the PCNA had been mutated to abolish such modifications. These results suggested that IR-IM related TLS occurs in the absence of PCNA K164 modification. Further analysis of a mutant simultaneously defective in SUMOylation and mono-ubiquitination (rad18 siz1) revealed that these modifications redundantly affected TLS as well as NHEJ. A genetic model based on these observations is proposed.     

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.