Identification and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene.

We have previously reported that a c-myc protein complex binds to the region upstream of the c-myc gene, where exist an origin of cellular DNA replication (ori) and a transcriptional enhancer. Both functions require a 21 bp long sequence, while the c-myc protein complex recognizes a 7 bp consensus therein. It was recently reported that single-stranded DNA binding proteins bound specifically to sequences that play roles in DNA replication or transcription. We examined for proteins binding to the single-stranded DNAs of the 21 bp element (myc(H-P)21). In a band shift assay with HL60 cells nuclear extract, probes of either the plus strand or the minus strand gave rise to specific signals. Mutation introduced within a short consensus (A/TCTA/TA/TT) present in both strands completely abolished binding in either case. Southwestern blotting analysis showed that proteins of molecular weight 105, 80, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either strand and 22 kDa to minus strand to the cognate A/TCTA/TA/TT consensus. These single-stranded DNA binding proteins were named MSSP, c-myc gene single strand binding proteins. We attempted to isolate the cDNAs encoding these proteins by screening a human cDNA library with the plus single-stranded oligonucleotide as a probe. Among several positive clones, we have characterized one, termed MSSP-1. MSSP-1 produced in E. coli as a fusion protein with GST specifically interacted with single-stranded TCTTAT (plus myc(H-P)21) and ACT-ATT (in minus myc(H-P)21), the consensus of which can be referred to as A/TCTA/TA/TT. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleoproteins. Interestingly, the MSSP-1/GST fusion protein specifically recognized myc(H-P)21 not only in single-stranded but also in double-stranded forms. Binding properties of MSSP-1 imply its functions in DNA replication. Furthermore, when the AT stretch in the SV40 ori core was substituted by TCTTAT, MSSP-1 promoted viral DNA replication depending on the consensus sequences.