XPF-ERCC1 is involved in various DNA repair pathways.8
XPF-ERCC1 is essential for NER and ICL repair. XPF-ERCC1 also plays a role in SSA,20, 21
one of the DSB repair pathways. Recently, it has also been demonstrated that XPF-ERCC1 functions in a Ku80-independent end-joining DSB repair pathway.22
In this report, we found an additional role of XPF-ERCC1 in ROS-induced DNA damage. The XPF-deficient rodent cell-line, UV41, displays cellular sensitivity to three ROS-generating reagents (H2
, paraquat, bleomycin) (). In vitro, XPF-ERCC1 removes a 3’-blocked end induced by ROS ( and ). These results show that XPF-ERCC1 may function in the repair of oxidative DNA damage by trimming a 3’blocked terminus induced by ROS. This putative activity would lead to the generation of a 3’-hydoxyl end that is accessible to a DNA repair polymerase to allow completion of SSB repair, BER and DSB repair.
Oxidative DNA damage, cyclopurines are implicated to be critical DNA lesions in the neurodegenerations observed in some XP patients.23
Cyclopurines are induced by γ-ray irradiation and other oxidative stress.24
Unlike other oxidative DNA damage, cyclopurines are shown to be repaired by NER, but not by BER.25
Because XPF-ERCC1 is an essential component of NER, cellular sensitivity of UV41 and UV20 to hydrogen peroxide could be a result of a defect of the repair of cyclopurines. However, XP-A
cells have been shown not to be sensitive to γ-ray irradiation, hydrogen peroxide and paraquat.12, 26, 27, 28
We are of the opinion that the observed sensitivity of UV41 to ROS-inducing chemicals is not due to a defect in NER activity.
The yeast counterpart of XPF-ERCC1, Rad1-Rad10, removes 3’-blocked termini and functions as a back-up enzyme of two major 3’-trimming enzymes Apn1 and Apn2.11
Inactivation of Rad1 (or Rad10) alone does not sensitize the cells to H2
The impact of Rad1-Rad10 on the repair of ROS-generated DNA damage can be seen only in the absence of Apn1 and Apn2.10, 11
Mammalian APN1 has also been proposed as a major enzyme to remove the 3’-blocked ends generated by ROS in SSB repair and BER.13, 29, 30
However, our results indicate that XPF-ERCC1 also plays a significant role in the processing of 3’-blocked ends. Many in vitro studies with APN1 included Mg2+
as a cofactor,13, 30
which is a poor co-factor for the trimming activity of XPF-ERCC1 (). XPF-ERCC1 might be involved in an APN1-independent SSB repair/BER pathway. Epistatic studies should be performed to determine the genetic relationship between XPF-ERCC1 and APN1 in the repair of ROS-induced DNA damage.
The roles of XPF-ERCC1 in the repair of “two-ended” DSBs induced by γ-ray or restriction enzymes have been reported. XPF-ERCC1 removes 3’-overhangs in a Ku86-independent end-joining repair pathway22
and also participates in single strand annealing (SSA) and gene conversion to repair DSBs induced by a restriction enzyme I-SceI
Intermediate DNA structures, which XPF-ERCC1 processes in these repair pathways, are believed to be a 3’-splay/flap-like structure. Hydrogen peroxide is also known to induce DSBs directly32
and the 3’-ends of these breaks can be blocked. These 3’-blocked ends can be removed from a 3’-splay/flap-like structure as a part of a 3’-overhang in an end-joining repair and SSA. In gene conversion to repair “two-ended” DSBs, DNA synthesis-dependent extension from one of the broken ends initiates the repair process.33
Our results indicate that XPF-ERCC1 can remove 3’-bloked ends at DSBs and therefore may participate in the repair of breaks generated directly by ROS and promote the initiation of repair synthesis.
A single strand break (SSB), if not repaired, results in the formation of a DSB after DNA replication. The resulting break is a “one-ended” DSB, which is distinct from the “two-ended” DSBs induced by γ-ray or restriction enzymes. “One-ended” DSBs are repaired by homologous recombination and required a 3’-hydroxy moiety at the broken end to initiate the repair process.34
If a SSB with a 3’-blocked end is located in the template for a lagging strand synthesis, a structure of the broken end could be a 3’-splay-like structure. This structure is a favorite for XPF-ERCC1, thus a single-stranded tail with a blocked end can be removed by the endonuclease activity of XPF-ERCC1. A SSB with a 3’-blocked terminus on a leading strand template will generate a gap structure that is similar to our DS/SS substrate. It is shown that XPF-ERCC1 and yeast Rad1-Rad10 are involved in the repair of camptothecin-induced Top1-lesions in a TDP1-independent pathway.35, 36
Camptothecin induces an abortive Top1-DNA complex at a 3’-terminus of a single strand nick. These Top1-lesions are normally repaired by TDP1-mediated single strand break repair.1
Unrepaired Top1-lesions can be converted to DSBs during DNA replication. In the absence of TDP1, XPF-ERCC1 and Rad1-Rad10 are proposed to remove Top1-lesions from the 3’-terminus of DNA and the DSBs are repaired by homologous recombination. XPF-ERCC1 might play a specific role in the repair of SSB-induced DSBs and/or gaps with 3’-blocked termini in conjunction with DNA replication.
XPF-ERCC1 is a structure-specific endonucelase that prefers a 3’-splay/flap structure with a 3’-single-strand tail.18
XPF-ERCC1 makes an incision in the double stranded DNA 3–5 nt from the junction of double stranded and 3’-single stranded DNA. Our results showed that XPF-ERCC1 makes an incision 5–7 nt from the 3’-ends, with or without damage (). Rad1-Rad10 removes 3’-blocked ends by a 3’-5’ exonuclease activity without showing a preference to damaged ends over non-damaged ends.11
XPF-ERCC1 also contains an intrinsic 3’-5’exonuclease activity,37
thus this exonuclease activity could remove 3’-blocked ends. Our time course experiments showed that XPF-ERCC1 makes an incision on DS/SS and DS substrates differently, but with a similar efficiency (Figs. S-2 and S-3
). An exonuclease normally degrades substrate DNA from an end, regardless of its structure, in the same fashion. It is likely that XPF-ERCC1 removes 3’-blocked ends by its endonuclease activity.
XPF-ERCC1 is one of the six factors that are essential for in vitro NER,38
XPF-deficient cell lines derived from XP patients are moderately sensitive to UV irradiation due to residual NER activities. It was reported recently that one patient with a mutation in XPF
displayed accelerated aging symptoms, but not the XP symptoms.14
A cell line derived from this patient (XFE cells) showed hypersensitivity to UV irradiation and DNA cross-linking agents due to a severe defect of XPF-ERCC1. It is also important to note that the XFE cells showed sensitivity to IR.22
Thus, XFE would also be defective in the repair of ROS-induced DNA damage. Accumulation of DNA damage has been long proposed to be one of the causes of aging. Because other XP patients do not show aging phenotypes, DNA repair defects in addition to the NER defect contribute to the aging phenotypes. An accumulation of various types of DNA damage, including ROS-induced DNA damage due to defects in multiple XPF-ERCC1-mediated DNA repair pathways, could contribute to the aging phenotypes observed in the XFE patient.