Novel roles of DNA damage repair enzymes in the processing of modified ribonucleotides embedded in DNA

From data in literature, it has well known that 100 million rNMPs are transiently present in mammalian DNA for several reasons. Moreover, the presence of RNA as single or more ribonucleotides into DNA results very dangerous for the cell because able to distort the double helix DNA. A specific pathway acts in order to remove this lesion called Ribonucleotide Excision Repair (RER) pathway in which RNase H2 has an important role as endonuclease, able to cleave at the 5’ side of rNMP in DNA. Although in last decade, huge steps forward have been done in this field, more studies are needed for better understanding the impact of this lesion on DNA and their back-up repair mechanism when RER does not work, as happens in several pathologies including cancer and Aicardi-Goutieres syndrome. Because of the high abundance of rNMPs in DNA and the ability of several polymerases to insert and elongate oxidized rGMP during DNA replication, the hypothesis of the incorporation of modified rNMPs (including abasic and oxidized rNMPs such as 7,8-dihydro-8-oxo-riboguanosine) results feasible. Moreover, these data underscore the possibility, not only regarding the presence of modified rNMPs into DNA, but also the necessity to determine how cells can target and remove oxidized or abasic rNMPs from DNA. Until now, nothing is known about the putative role of Base Excision Repair (BER) pathway in the removal of unmodified and modified rNMPs embedded in DNA. APE1 is the most important protein that works in BER pathway as endonuclease, able to cleave 5′ side of deoxyabasic sites generated spontaneously or following the processing of glycosylases (including OGG1) on a damaged (including oxidized) base. Moreover, APE1 has nucleotide incision repair activity on different modified bases, which are directly repaired by APE1 bypassing the action of specific glycosylases. Finally BER, but specifically APE1, has an important involvement in RNA metabolism and RNA-decay as demonstrated by its ability to cleave abasic single-stranded RNA and for having 3′-RNA phosphatase and weak 3′-5′ exoribonuclease activities. For this reason, there is a high likelihood that BER enzymes could be involved in the processing of rNMPs in DNA, particularly in the case of chemically modified rNMPs, such as abasic and oxidized rNMPs. Therefore, we focused our attention on enzymes belonging to RER and BER enzymes in order to investigate: If BER can be work as a back up repair mechanism when RER is inefficient; If RER is involved in the processing of abasic and oxidized rNMPs embedded in DNA; and if not, if BER is involved in the processing of abasic and oxidized rNMPs embedded in DNA. For all the experiments, we have used different DNA oligonucleotides in which an abasic or an oxidized ribonucleotide was embedded in the middle of the sequence. First, we tested recombinant and cell-free extracts RNase H2. Surprisingly, we discovered that RNase H2, although able to process normal rG embedded in DNA, is not able to cleave abasic and oxidized rNMPs embedded in DNA leaving the hypothesis that RER can not work on this lesion. After that, we moved on BER pathway, specifically on APE1 and OGG1 proteins. We discovered that APE1, as 5’-endonuclease, does not cleave unmodified rNMPs embedded in DNA. So, APE1 may not work as back up when RNase H2 is inefficient as happens for different pathologies including cancer and Aicardi-Goutieres syndrome. We discovered and characterized an unknown APE1 activity on abasic ribonucleotide embedded in DNA. We then analyzed the activities of 8-oxoguanine DNA glycosylase (OGG1) and APE1 to recognize and cleave an oxidized rNMP. Our data demonstrate that OGG1, although able to bind the oxidized rG, is inefficient in the cleavage of this substrate. Surprisingly, APE1 shows a weak endoribonuclease activity on the oxidized substrate, maybe associable to a putative NIR activity of APE1. All these results show a strong impact on the DNA repair field.