Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex

Abstract

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

Figure 1

Mammalian SMC5 and SMC6 proteins. (A) Schematic of the hSMC5 and hSMC6 proteins showing the conserved Walker A nucleotide-binding motif and the Walker B motif in red and green, respectively, and the bipartite nuclear localization motif in blue. The underlines represent the regions of protein against which the indicated antibodies were increased. (B) Genomic localization of the mSMC6 gene. Double stain with the use of Texas Red-mSMC6 DNA (red) and an FITC-labeled chromosome 12 telomere-specific probe (green). (C and D) Amino acid sequence alignments between the hSMC6 and hSMC5 N-terminal (C) and C-terminal (D) regions and published SMC protein sequences. Identical amino acids are highlighted in black and conserved residues are indicated in gray.

Figure 2

Anti-hSMC5 and anti-hSMC6 antibodies and their use in expression studies. (A) Western blots with three different anti-hSMC6 antibodies on human 1BR.3Neo cell extract (lanes 1, 4, and 7), in vitro translated ΔN-hSMC5 (lanes 2, 5, and 8) and in vitro translated hSMC6 (lanes 3, 6, and 9). (B) Western blots with two different anti-hSMC5 antibodies on 1BR.3Neo cell extract (lanes 1 and 4), in vitro translated ΔN-hSMC5 (lanes 2 and 5), and in vitro translated hSMC6 (lanes 3 and 6). (C) 1BR.3Neo cell extracts were prepared in the presence (lane 1) or absence (lane 2) of phosphatase inhibitors or were treated with alkaline phosphatase (lane 3) before immunoblotting with 6B2 anti-hSMC6 antibody. (D) Western blot of 1BR primary fibroblast cell extracts (50 μg per lane) after γ-irradiation (5 Gy). Blots were probed with anti-hSMC6 (6B2), anti-tubulin, and anti-p53 antibodies. (E) Western blot of cell extracts prepared after the release of 1BR.3Neo-transformed fibroblasts from a thymidine-mimosine cell cycle block. Blots were probed with the 6B2 anti-hSMC6 antibody and ku80 antibody (as a loading control).

Figure 3

Interaction of hSMC5 and hSMC6. Human cell extracts were immunoprecipitated with preimmune (PI), 6A1, or 5A1 antisera as indicated and immunoblotted with 6B1 anti-hSMC6 (left) or 5A1 anti-hSMC5 (right) antibody. (B) As in A but with in vitro translated hSMC6 or ΔN-hSMC5 in place of human cell extracts.

Figure 4

Subcellular localization of hSMC5 and hSMC6. (A) Nuclear and cytoplasmic extracts of human 1BR.3Neo cells containing 50 μg of protein were electrophoresed and immunoblotted with the indicated antibodies. 6B2 was used for hSMC6 and 5B1 for hSMC5. (B) Nuclei were extracted for 1 h at 4°C with the indicated concentrations of NaCl or for 1 h at 25°C with DNAse I at the indicated units/milliliter. The nuclei were then centrifuged and the amount of hSMC6 in supernatant (S) and pellet (P) were determined by immunoblotting with 6B1 anti-hSMC6. (C) Nuclei were extracted for 1 h at 4°C in the presence or absence of phosphatase inhibitors, without treatment, with 2 M NaCl, or with 2 M NaCl and 2.5% Triton X-100. After centrifugation, the amounts of the indicated proteins in the supernatant (S) and pellet (P) fractions were determined by immunoblotting. Antibodies used were 6B2 (top panel), 6B1 (second panel), 5B1 (third panel), anti-PCNA (fourth panel), and anti-lamin B (fifth panel).

Figure 5

Immunofluorescence studies of hSMC6. (A) Human 1BR.3Neo fibroblasts were stained with 6B1 anti-hSMC6. (B) HTC75 cells were transfected with FLAG-tagged hSMC6 and then stained with anti-FLAG M5 antibody. (C and D) A mitotic cell stained with 6B1 (C) or 4′,6-diamidino-2-phenylindole (D). (E–I) Cells fixed with 3.7% formaldehyde (E), 30% methanol/70% acetone (F), or pre-extracted with 0.5% Triton X-100 before formaldehyde fixation (G) were stained with 6B2 anti-hSMC6. The cells shown in G were counterstained with anti-SC35 splicing factor antibody (H). The overlay image of the two antibodies is presented in I. Bar, 10 μm.

Figure 6

Tissue-specific expression of hSMC6 and hSMC5. (A) Northern blots of 3 μg of mRNA prepared from proliferating 1BR.3Neo-transformed human fibroblasts hybridized with hSMC6 and hSMC5 probes. (B) Human tissue-specific Northern blot (Clontech) probed for the indicated mRNAs.

Figure 7

Meiotic prophase localization of hSMC5 and hSMC6. (A) A midpachytene mouse spermatocyte immunostained with antibodies to chromosome cores and with 6B1 anti-hSMC6 antibody. (B) An early diplotene spermatocyte stained with anti-core antibody and 6B1. The sex vesicle and sex chromosome cores are recognized by the anti-hSMC6 antibody. (C and D) Midpachytene (C) and early diplotene (D) spermatocytes stained with anti-COR1 and 5A1 anti-hSMC5 antibody. The X-chromosome (X), Y-chromosome (Y), sex vesicle (SV), and synaptonemal complexes (SC) are indicated. Bar, 10 μm.