DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair

Abstract

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

Figure 1

Organization of human MutS homolog genes. The MSH3 gene is divergently transcribed from a promoter region shared with the DHFR gene (11, 41). The DHFR copy number (n) is about 200 in Mtx^r HL-60R cells (11). Genes encoding the two polypeptides that comprise hMutSα, MSH2 and MSH6, reside in close proximity on chromosome 2 (20, 33). The relative orientation of transcription shown for MSH2 and MSH6 is arbitrary.

Figure 2

Western analysis of MutS homologs. (A) In each of the first four lanes, 75 μg of the indicated nuclear extract was loaded and probed independently for hMSH2, hMSH3, or hMSH6. In the latter four lanes, the indicated amount (ng) of purified hMutSα or hMutSβ was loaded to demonstrate the lower detection limits of the experiment. (B) The efficiency of retention of hMSH2 on single-stranded DNA cellulose is shown for extracts derived from HL-60 and HL-60R cells. The volume loaded was normalized in each case to that of the extract sample. In the two right lanes, purified hMutSα (100 ng) was subjected to electrophoresis in the presence or absence of LoVo extract (50 μg) to demonstrate the effect of high protein concentration on electrophoretic mobility and blotting efficiency.

Figure 3

Resolution of hMutSα and hMutSβ on MonoQ. MonoQ anion exchange FPLC chromatography was performed on DNA cellulose eluates (see Materials and Methods) prepared from HL-60 extract (Upper) or HL-60R extract (Lower). In each case, the upper section shows results of a Coomassie-stained gel, and the middle section shows the corresponding Western blot for the hMSH2, hMSH3, and hMSH6 polypeptides. The positions of subunit polypeptides of hMutSα and hMutSβ in stained gels are indicated by arrows. Each fraction (2.5 μl of 500 μL) was tested for its ability to complement repair-deficient LoVo extract on a 3′ _/CA_/ insertion–deletion heteroduplex.

Figure 4

Complementation activity of hMutSα and hMutSβ. Circular heteroduplexes containing a G-T base–base mismatch (Upper) or a _/CA_/ insertion–deletion mispair (Lower) were used to determine extract repair activities in the absence of exogenous proteins (empty bars), in the presence of added hMutSα (hatched bars), or hMutSβ (filled bars). Error bars are ± 1 SD. The G-T substrate had a strand break at the Sau96I site located 181 nucleotides 5′ to the mismatch (shorter path in the circular substrate), whereas the _/CA_/ heteroduplex had a strand break 125 nucleotides 3′ to mispair (23, 42).