DNA repair inventory
Comparison of DNA repair proteins in S. cerevisiae to E. cuniculi's genome and proteome via BLAST and PSI-BLAST searches has revealed that E. cuniculi appears to contain a reduced set of proteins in all major repair pathways. Of the 56 repair genes that were sought in E. cuniculi, 16 are absent, with another 6 potentially absent. Six out of 14 DNA polymerases or polymerase subunits are absent (See Table ). Although all repair pathways have been reduced, the loss of genes is not distributed evenly among pathways. Each process has been affected differently by genome reduction. A detailed discussion of the components of each pathway is presented below.
S. cerevisiae DNA polymerases and proteins that participate in the five primary DNA repair pathways.
Base excision repair (BER)
BER is one of the least complex of the DNA repair mechanisms, and involves only a small number of proteins. When a base becomes damaged, it is recognized by a DNA glycosylase that is specific for the particular base and/or the type of damage (methylation, oxidation, etc.). S. cerevisiae
contains four types of glycosylases, although far more have been found in other organisms (animals, bacteria, etc.) [11
]. Glycosylases cleave the glycosylic bond between the base and the deoxyribose to remove the damaged base, at which point a non-specific apurinic/apyrimidinic (AP) endonuclease (Apn1 or Apn2) removes the remaining deoxyribose phosphate to create a gap [12
]. In short patch BER (which replaces a single nucleotide), the gap is filled by DNA polymerase β. In long patch BER (which replaces two or more nucleotides), the DNA polymerases β, or δ and ε in concert with proliferating cell nuclear antigen (PCNA) synthesize several nucleotides which displace the original DNA strand. Rad27 then removes the displaced DNA. The ligase Cdc9 (or DNA ligase III and Xrcc1 in other eukaryotes) is used to seal the nick [13
The Rad1-Rad10 and Mus81-Mms4 endonucleases are also believed to play minor roles in BER by processing the 3' ends of the DNA once an incision has been made into the sugar-phosphate backbone [12
's BER pathway appears to be nearly complete, but lacks DNA polymerase β, the Cdc9 DNA ligase (but possesses Xrcc1, the cofactor of the ligase used in this process in some eukaryotes) and part of a 3' endonuclease, Mms4. (See Table ) Deletion of either polymerase β or Mms4 is not a lethal mutation in yeast, however S. cerevisiae
cannot survive in the absence of Cdc9 [15
]. Another ligase is likely utilized for BER in E. cuniculi
, as sharing non-specialized enzymes between pathways is not uncommon in S. cerevisiae
(see discussion), and there is no reason to believe that this is not the case in E. cuniculi
Nucleotide excision repair (NER)
NER is used primarily to remove bulky lesions from DNA, such as inter- and intra-strand crosslinks. NER is a more complex process than BER, and utilizes a large number of proteins that are evolutionarily conserved among eukaryotes. NER is comprised of two subpathways, global genome repair (GGR) and transcription-coupled repair (TCR). As is suggested by their names, the subpathways act on different types of DNA: DNA that is not transcribed (or the non-transcribed strands of expressed genes), and actively transcribed DNA, respectively. In both GGR and TCR, DNA damage recognition is the first step to occur, followed by DNA unwinding. Next, incisions are made on either side of the aberrant base(s), and a total of 25–30 nucleotides on either side are removed as a single strand. The gap is then filled by DNA polymerase and sealed by DNA ligase. Recruitment of five multi-protein complexes, nucleotide excision repair factors (NEFs) 1 through 4 and the replication protein A (RPA) complex, is believed to take place in a stepwise manner to complete this process. The RPA complex is composed of Rpa1 and Rpa2 and recognizes damaged DNA.
In GGR, the first protein complex to arrive at the damaged site is NEF4, which recognizes damage and is composed of the proteins Rad7 and Rad16. Rad7 binds the NEF2 complex (Rad4/Rad23), recruiting it to the damaged site and increasing DNA binding efficiency. The presence of NEF4 is not strictly required for the recruitment of NEF2 to the DNA lesion, but facilitates the process. The above proteins do not act in the other sub-pathway; in TCR, initiation of repair takes place when an RNA polymerase stalls. Two proteins involved specifically in TCR, Rad26 and Rad28, also participate in the beginning of this process [16
In both GGR and TCR, NEF1 and NEF3 are the next components to be recruited, and are held at the damage site by NEF2. NEF1 is composed of Rad1, Rad10 and Rad14, while NEF3 is composed of Rad2 and transcription elongation factor IIH (TFIIH). TFIIH contains the Rad3, Rad25, SSL1, TFB1, TFB2 and TFB3 proteins, and provides the single strand DNA helicases required for repair proteins to access the damaged site. Rad1 and Rad10 form a heterodimer that acts as a single strand endonuclease at the 5' end of the stretch of damaged DNA and Rad2 is a single strand endonuclease that cuts at the 3' end. RPA is thought to be the last player to arrive at the scene.
Many of these proteins also have roles in other cellular processes, such as recombination and transcription, therefore mutants express defects in several pathways. For a comprehensive review of NER, see Prakash and Prakash [17
Most proteins participating in NER are present in E. cuniculi
, with two exceptions. Half of a GGR heterodimeric damage sensor complex (Rad23) and the Tfb1 subunit of TFIIH appear to be absent (See Table ). Rad23 appears to have diverse functions within the cell, ranging from DNA repair to the regulation of a cell-cycle checkpoint and protein degradation. Specifically, this protein helps to prevent the degradation of Rad4, as well as serving a role with the 26S proteasome in regulating the NER pathway [18
]. Deletion of Tfb1 in S. cerevisiae
is lethal [20
], likely due to loss of function in transcription.
The presence or absence of Rad7 and Rad16 were not confirmed, as BLAST and PSI-BLAST searches using S. cerevisiae and S. pombe sequences as queries did not return homologues from most animals or other eukaryotes besides fungi.
Methyltransferases are present in both eukaryotes and prokaryotes and remove certain DNA lesions involving methylation (O6
-methylthymine). These proteins irreversibly relocate methyl groups from DNA to their own cysteine residues, and are therefore suicide enzymes [21
does not possess the methyltransferase found in other eukaryotes, Mgt1. Deletion of this gene is not lethal in S. cerevisiae
Mismatch repair (MMR)
In MMR, mismatches are recognized by the heterodimers MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3). Single base mismatches are recognized by MutSα and insertion/deletion loops (IDLs) less than about 9 nucleotides in length are recognized by MutSβ [22
]. Both MutSα and MutSβ can recognize a single unpaired nucleotide. PCNA is also involved in MMR, perhaps assisting in these initial recognition steps. MutLα (Mlh1/Pms1) binds MutSα and β and allows them to efficiently bind to IDLs and mismatches. The exonuclease Exo1 then excises the mismatched base(s) and a DNA polymerase and DNA ligase fill and seal the gap.
It should be noted that the proteins required for the MMR process differ among eukaryotes. For instance, Drosophila
lack Msh3 homologues, and therefore do not require them for the removal of IDLs [22
does possess a Msh3 homologue, but it appears to play a different role within the cell, instead participating in recombination [23
The majority of S. cerevisiae
MMR proteins are present in E. cuniculi
. The sole missing protein is Msh3, a subunit of the MutSβ heterodimer that recognizes small IDLs (See Table ). Deletion of this gene in S. cerevisiae
is not lethal (See discussion) [20
Homologous recombination repair (HRR)
HRR is the major form of double strand break repair utilized in yeast. A double stranded break is recognized by damage recognition proteins, and single stranded overhangs are generated at both sides of the break. A region of the genome that is homologous to the single stranded overhangs is then found. Strand invasion follows, and the homologous (non-damaged) DNA is used as a template for synthesis on the broken strand. HRR is completed through re-annealing of the broken DNA strand and ligation. See figure for an overview of this process.
Figure 2 The homologous recombination repair pathway. (See text for explanation.) Blue proteins are present in E. cuniculi; all others are absent. Newly synthesized DNA is indicated in grey. Although the MRX complex (Mre11/Rad50/Xrs2) acts in damage recognition (more ...)
The Rad51, 52, 54, 55 and 57 proteins perform most steps of the HRR process. Rad51 is a homologue of the bacterial enzyme RecA, and is well conserved within eukaryotes. When a double strand break is formed, the MRX complex (which is composed of Mre11, Rad50 and Xrs2, and also acts in NHEJ) is involved in damage recognition. The DNA ends on either side of the break are then chewed back in the 5' to 3' direction by an unknown nuclease. Rad24 (which is a checkpoint protein as well) is also involved in end processing. The results of this process are short 3' overhangs on either strand. RPA (which also acts in NER, as described above) then coats the overhangs. RPA is later replaced by Rad51, with the aid of Rad52, Rad55/Rad57, and very likely Rad54 as a genome-wide search for homologous sequences takes place. Strand invasion then occurs while the helicase Hpr5 removes Rad51 from the DNA. DNA is synthesized by an undetermined polymerase based on the donor template strands, and then ligated. Although the mechanism is not clear, it is evident that the Rad55/Rad57 complex is somehow involved in this last step. The Sgs1 helicase plays a specific role in the repair of double strand breaks generated by the stalling of a replication fork. For a review of HRR, see Aylon and Kupiec [24
Three other signaling and damage sensor proteins are also involved in the HRR pathway, as well as the BER pathway and the NHEJ pathways. The Rad17/Med3/Ddc1 (9-1-1) complex triggers DNA damage checkpoints [25
], and stimulates repair pathways [26
] as well as various individual repair proteins, including DNA polymerase β [27
], Rad51 [28
] (HRR), Rad27 [29
] (BER, NHEJ) and Cdc9 [30
lacks more than half of the proteins involved in the HRR pathway. Almost all steps of the process are affected by these losses (see discussion). Missing proteins include the Hpr5 helicase, Rad54 and Rdh54 (See Figure , Table ). Rad24 and the 9-1-1 complex are all absent from the cell signaling pathways. S. cerevisiae
single mutants lacking these proteins are viable [20
], likely due to yeast's ability to use either double strand break repair pathway (HRR or NHEJ) to fix damaged DNA.
The presence or absence of Rad55 and Rad57 was not determined. Rad55 and Rad57 are paralogs of Rad51. PSI-BLAST searches using S. cerevisiae Rad55 and Rad57 proteins retrieve Rad51 in other fungi, therefore making it difficult to discern the presence of these proteins in E. cuniculi, which is related to fungi.
Non-homologous end joining repair (NHEJ)
NHEJ is the second form of double strand break repair that is a separate, though not completely independent pathway from HRR. In S. cerevisiae
this method of double strand break repair plays a minor role compared to the HRR pathway. Upon double strand break formation, damage is recognized and both ends of the lesion are brought together through the action of several proteins. A minimal amount of DNA synthesis occurs, which is followed by ligation. As DNA on either side of the break may be degenerated before the break is repaired, the potential for information loss in this case is substantial [24
The NHEJ process begins when the Ku complex (Ku70/Ku80) binds either end of the double strand break (See Fig ). These proteins are DNA-dependent protein kinases that also have a role in telomere maintenance. Once bound to the damaged site, the Ku complex is responsible for recruiting the MRX complex for the next stage in the repair process. The MRX complex is composed of Rad50 (an ATP binding protein), Mre11 (a 5'-3' exonuclease) and Xrs2 (responsible for aligning the MRX complex with the break site) [31
]. Dnl4/Lif1 (a DNA ligase complex) is tethered to the break site by Xrs2 and the Ku complex. The DNA polymerase Pol4 and the structure-specific nuclease Rad27 are the last players to arrive at the scene, thus completing the repair complex.
The non-homologous end joining repair pathway. (See text for explanation.) Blue proteins are present in E. cuniculi; all others are absent. Newly synthesized DNA is indicated in grey. (Modified from Hefferin and Tomkinson .)
All of the yeast NHEJ proteins are present in most eukaryotes, and the core of Ku70 and 80 is homologous to a smaller bacterial protein that performs the same function, thus indicating a large degree of conservation. For a review of this process, see Hefferin and Tomkinson [32
is missing nearly all NHEJ proteins. Absent proteins include Ku70, Ku80, Xrs2, Dnl4, and Pol4 (See Fig , Table ). As is the case with single S. cerevisiae
mutants for genes involved in the HRR pathway, most are viable [20
] due to yeast's ability to rely on the other (HRR) double strand break repair pathway.
Although there are animal homologues of Lif1 and Xrs2 (Xrcc4 and Nbs1, respectively), BLASTP and PSI-BLAST searches using yeast proteins did not retrieve homologues in any organisms other than fungi. The presence or absence of these proteins is therefore not known.
DNA polymerases are essential for both genome replication and repair. There are several polymerases present in eukaryotic cells, all of which serve particular functions within the cell. The polymerases α, δ and ε act in the process of genome replication, but also play roles in certain repair processes, notably NER and HRR. Polymerase γ acts solely within mitochondria, while all other polymerases are nuclear. Polymerase β is a specialized repair polymerase that is involved in BER and NHEJ. The polymerases ζ, η and Rev1 help prevent double stranded DNA breaks from forming during replication due to their ability to synthesize DNA through a lesion, where polymerases α, δ and ε stall and dissociate from the replication fork [4
Of the 8 polymerases identified in S. cerevisiae
that have human counterparts (confirming that they are not fungal or ascomycete specific), E. cuniculi
possesses 3: α, δ and ε (See Table ). All three of these polymerases are necessary for viability in S. cerevisiae
. All of the polymerases that are absent in E. cuniculi
are utilized solely for repair or lesion bypass and are not essential for viability, likely because their function is replaced by other polymerases [20