The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD

The human Rad9 checkpoint protein is a subunit of the heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex that plays a role as a damage sensor in the DNA damage checkpoint response. Rad9 has been found to interact with several other proteins outside the context of the 9-1-1 complex with no obvious checkpoint functions. During our studies on the 9-1-1 complex, we found that Rad9 immunoprecipitates contained a 240 kDa protein that was identified as carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Further investigations revealed that only free Rad9, but not Rad9 within the 9-1-1 complex, bound to CAD. The rate-limiting step in de novo pyrimidine nucleotide synthesis is catalyzed by the carbamoyl phosphate synthetase II (CPSase) domain of CAD. We find that Rad9 binds to the CPSase domain, and, moreover, this binding results in a 2-fold stimulation of the CPSase activity of CAD. Similar results were also obtained with an N-terminal Rad9 fragment. These findings suggest that Rad9 may play a role in ribonucleotide biosynthesis.     

Human and E.coli excinucleases are affected differently by the sequence context of acetylaminofluorene-guanine adduct.

Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.     

Formation and function of flavin anion radical in cryptochrome 1 blue-light photoreceptor of monarch butterfly

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.     

Use of an Escherichia coli mutant strain permits measurement of single-stranded apurinic-apyrimidinic endonuclease in crude extracts: studies with untransformed cells and cells transformed with plasmids containing the uvrC gene.

We have constructed a strain of Escherichia coli that is defective in exonuclease VII and uracil-DNA glycosylase activities. This strain (xse ung) facilitates the quantitation of single-stranded apurinic-apyrimidinic endonuclease activity in crude extracts. Quantitative comparisons of single-stranded apurinic-apyrimidinic endonuclease activity under conditions in which uvrC protein is overexpressed showed no differences, suggesting that single-stranded apurinic-apyrimidinic endonuclease and uvrC protein are probably distinct.     

Active site of (A)BC excinuclease. I. Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues.

(A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base. The activity results from the ordered action of UvrA, UvrB, and UvrC proteins. The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC. To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro. The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated. Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity. Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity. Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.     

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (pol β), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol β is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol β or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol β with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a “hit and run” mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.The mammalian genome experiences constant stress from both external and internal factors that causes genomic instability. Eukaryotic cells have developed a number of DNA repair pathways that correct DNA damage before it results in permanent chromosomal alteration. Base excision repair (BER)³ is the major pathway responsible for reversing DNA damage sustained by individual nucleotide bases. Mammalian BER is initiated by DNA glycosylases, which recognize structural alteration of a nitrogenous base and excise it leaving an intact sugar-phosphate backbone with an apurinic/apyrimidinic (AP) site (1). AP sites in humans are detected by AP endonuclease 1 (APE1) that cleaves the phosphate backbone of the damaged strand, leaving a nick with a 3′-OH group and a 5′-deoxyribose phosphate (dRP) residue. The dRP-bordered nick is not a substrate for ligation. If the dRP residue is not oxidized or reduced, repair can proceed via a short patch-BER pathway, in which the dRP residue is removed by the 5′-lyase activity of DNA polymerase β (pol β), which concurrently fills in the 1-nt gap, and the resulting nick is sealed by the DNA ligase III-XRCC1 complex (2-4).However, if the oxidative state of the dRP is altered, the lyase activity of pol β is inhibited, but the polymerase activity of pol β can still displace the oxidized or reduced dRP residue into a 2-10-nt 5′ flap intermediate, which will then be cleaved by FEN1 and subsequently joined by LigI (4-7). This process is known as long patch-base excision repair (LP-BER). Recent studies examining the relevance of the two different pathways in vitro predict a predominant role for short patch-BER in the cell as compared with LP-BER (8). Because the cell undergoes constant repair of damaged bases, it is very difficult to assess the relative use of one pathway over the other in vivo. Studies using plasmid DNA containing defined DNA damage have been used as an indirect approach to evaluate the role of the two different BER pathways in cells and the size of the DNA repair patches (9). Results from these studies have shown that repair patches of 6-12 nucleotides are generated during repair of plasmids that contain a single base lesion, at least supporting the existence of LP-BER in vivo.LP-BER has also been proposed to proceed by either a PCNA-dependent sub-pathway involving the use of DNA pol δ/ε or a PCNA-independent sub-pathway that uses only DNA pol β. However, most LP-BER reconstitution experiments in vitro indicate that pol β works more efficiently than pol δ with the other proposed LP-BER proteins. FEN1 is known to stimulate pol β-mediated DNA synthesis on an LP-BER substrate suggesting that these two proteins interact functionally and mechanistically (10). pol β has also been shown to interact with LigI by co-immunoprecipitation experiments indicating that they might be a part of a multiprotein DNA repair complex (11).The heterotrimeric protein complex, Rad9, Rad1, and Hus1 (the 9-1-1 complex), plays a significant role in the early recognition of DNA damage and recruiting appropriate proteins to repair sites. The 9-1-1 complex interacts with several of the proteins involved in the proposed BER pathways, including DNA glycosylases (12-14), APE1 (15), pol β (16), FEN1 (17,18), and LigI (19, 20). In a recent report (15), the 9-1-1 complex was shown to interact both physically and functionally with APE1 and pol β and to stimulate their respective activities. Stimulation of the endonuclease ensures the abasic site is recognized and cleaved off efficiently. Stimulation of nucleotide addition by pol β is expected to promote the LP-BER sub-pathway, as 9-1-1 stimulates the strand displacement activity of pol β, thereby requiring FEN1 flap cleavage before ligation to repair the site of damage. Because 9-1-1 is structurally similar to the sliding clamp PCNA, early studies were focused on determining the effects of 9-1-1 on DNA replication and repair proteins previously shown to be stimulated by PCNA. The 9-1-1 complex has been reported to stimulate both FEN1 cleavage (17, 18) and nick sealing by LigI (20) in vitro. However, the 9-1-1 clamp poorly stimulated FEN1 and LigI in the entire LP-BER-reconstituted system as compared with strong stimulation by 9-1-1 of individual cognate substrates (15). The authors (15) suggest that FEN1 and LigI evolved to respond to stimulation by PCNA and not 9-1-1 during LP-BER. The issue with this explanation is that it does not take into consideration how LP-BER would be efficiently carried out when damage-induced p21 binds and inhibits PCNA (21).To define how 9-1-1 interacts with the components of BER, we have reconstituted the entire LP-BER pathway using purified human enzymes and substrates that simulate an abasic site created after recognition and cleavage of damaged base by a glycosylase. Similar to results of Gembka et al. (15), we observe much less stimulation of either FEN1 or LigI by 9-1-1 in the fully reconstituted system compared with 9-1-1 stimulation of FEN1 on a flap substrate or LigI on a nicked substrate alone. Our subsequent analysis of the protein-protein interactions among the various LP-BER enzymes provides insight into why the 9-1-1 clamp exhibits minimal stimulation in the reconstituted system. Moreover, our mechanistic characterization of the significant role of pol β in mediating the activities of various enzymes in the multiprotein repair complex both explains the behavior of 9-1-1 and strongly suggests the existence of the LP-BER pathway in vivo.