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 D NA replication (ori) and a transcriptional enhancer. Both functions re quire a 21 bp long sequence, while the c-myc protein complex recognize s a 7 bp consensus therein. It was recently reported that single-stran ded DNA binding proteins bound specifically to sequences that play rol es in DNA replication or transcription. We examined for proteins bindi ng 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 th e plus strand or the minus strand gave rise to specific signals. Mutat ion introduced within a short consensus (A/(T)CT(A)/T(A)/(T)T) present in both strands completely abolished binding in either case. Southwes tern blotting analysis showed that proteins of molecular weight 105, 8 0, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either s trand and 22 kDa to minus strand to the cognate A/(T)CT(A)/(T)A/(T)T c onsensus. 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 pos itive clones, we have characterized one, termed MSSP-1. MSSP-1 produce d 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/(T)CT(A)/T(A)/(T )T. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleo proteins. Interestingly, the MSSP-1/GST fusion protein specifically re cognized myc(H-P)21 not only in single-stranded but also in double-str anded forms. Binding properties of MSSP-1 imply its functions in DNA r eplication. 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.