Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.

transfection. After incubation for 1 hour, the medium was replaced with new medium containing nucleoside. The cultures were subjected to cell proliferation assays after incubation for a further 1, 2, 3, or 4 days.

Establishment of an MLH1-proficient H414 cell line.
A human MLH1-proficient H414 cell line was established by targeted knock-in of wild-type exon 9 sequence into one allele of human MLH1 in HCT116 cells. A 2.3 kbp DNA region including exon 7 and 8 of MLH1 was amplified as a 5'-targeting element by PCR from genomic DNA of HeLa cells. SalI and NotI recognition sites were added to its 5' and 3' ends, respectively, using a primer set for 5'-targeting element (Supplementary Table S3).
Similarly, a 3.6 kbp DNA region including normal exon 9 of MLH1 was amplified as a 3'-targeting element by PCR from HeLa cells, and NotI and AscI sites were added to its 5' and 3' ends, respectively, using a primer set for 3'-targeting element. A promoter-less Neo expression cassette (IRES-Neo-pA) was amplified from pIRESneo2 (Clontech, Mountain View, CA, USA), and a NotI site and a loxP sequence, ATAACTTCGTATAGCATACATTATACGAAGTTAT, were added to both 5' and 3' ends of the Neo cassette. These DNA fragments were assembled in pBluescript II KS with a MC1DT-ApA cassette as described previously (Supplementary Figure S3A) 3 . The targeting vector was linearized by AscI digestion and transfected into HCT116 cells by electroporation. For the selection of targeted cells, G418 was added to culture medium at 350 µg/ml. After 14 days, colonies were isolated and expanded. Genomic DNA from individual colonies was screened for gene targeting by PCR using the primer sets, P1-F/ P1-R and P2-F/ P2-R, and two targeted clones (clone 1 and 2) were obtained. We transfected a CRE recombinase expressing plasmid, pCXCre (a kind gift from Dr. Masaru Okabe, Osaka University), into these clones to delete the floxed Neo cassette. The transfected cells were cultured in medium with or without G418. A G418-sensitive stable clone derived from clone 1 was screened to be MLH1-proficient by sequence analysis of MLH1 exon 9, and by western blot analysis of MLH1 protein with a mouse anti-MLH1 antibody (Supplementary Figure S3B and C). We termed this clone, H414.
Preparation of primary MEFs. Itpa and Mlh1 KO mice were previously established as described 4,5 .
Pregnant female mice were euthanized by cervical dislocation, and then the embryos were collected.
Genotypes were analysed by PCR using tail DNA. PCR primers to detect Itpa-mutant, Mlh1-mutant, and wild-type alleles are described in Supplementary Table S3. Skin fibroblasts were aseptically isolated from at least five independent embryos of each genotype. Their genotypes are shown in Supplementary Table S4. These primary MEFs were cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37°C in a 5% CO 2 atmosphere. Primary MEFs were harvested by treatment with 0.15% trypsin-0.08% EDTA in phosphate-buffered saline and reseeded for further passage. Those from Passage 2 were stored in liquid nitrogen as primary MEFs.
The preparation of nuclear DNA from these samples was performed using a DNA Extractor ® TIS Kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions, except that 10 mM 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO, Wako) and 20 µM 2'-deoxycoformycin, an adenosine deaminase inhibitor (Santa Cruz), were added to all reagents at all stages of manipulation, according to the method described by Taghizadeh et al. 6 The DNA samples were digested with Nuclease P1 (Wako) and acid phosphatase from potato (Sigma-Aldrich) in the presence of 20 mM TEMPO and 20 µM 2'-deoxycoformycin, and the digested samples were subjected to LC-MS/MS analysis using the Shimadzu VP-10 HPLC system (Shimadzu Corporation, Kyoto, Japan) connected to the API3000 MS/MS system (PE-SCIEX, Applied Biosystems), as described previously 7 . To analyze dI in DNA from H414 cells, we exactly followed Taghizadeh's protocol 6 and used Shimazu Nexera  (Table S3). The PCR products were analyzed for their sensitivity to