Fig 5.

BRLF1 binds to unmethylated and methylated DNA similarly in vitro. BRLF1 binding to the unmethylated versus methylated forms of an RRE from the BMLF1 promoter (33) (A), two predicted RREs from the BALF2 promoter (B), and an RRE from the late BLRF2 promoter (33) (C) was measured by EMSAs performed with whole-cell extracts. Extracts were derived from HeLa cells transfected with a truncated mutant of BRLF1 (R550) or SG5 control vector. Anti-BRLF1 antibody was added to the indicated reaction mixtures to ensure that retarded probe was indeed bound by BRLF1. BRLF1-DNA complexes, as well as supershifted complexes, are designated by arrows. The underlined cytosines indicate methylated sites in each RRE sequence (shown below the respective EMSA image). Boxed nucleotides encompass the core binding site where BRLF1 directly contacts DNA. This sequence is separated by a 9-nucleotide spacer.