Related sites in human and herpesvirus DNA recognized by methylated DNA-binding protein from human placenta
X Y Zhang1, P C Supakar, R Khan
1Department of Biochemistry, Tulane Medical School, New Orleans, LA 70112.
Abstract:
Methylated DNA-binding protein (MDBP) from mammalian cells binds specifically to six pBR322 and M13mp8 DNA sequences but only when they are methylated at their CpG dinucleotide pairs. We cloned three high-affinity MDBP recognition sites from the human genome on the basis of their binding to MDBP. These showed much homology to the previously characterized prokaryotic sites. However, the human sites exhibited methylation-independent binding apparently because of the replacement of m5C residues with T residues. We also identified three other MDBP sites in the herpes simplex virus type 1 genome, two of which require in vitro CpG methylation for binding and are in the upstream regions of viral genes. A comparison of MDBP sites leads to the following partially symmetrical consensus sequence for MDBP recognition sites: 5'-R T m5Y R Y Y A m5Y R G m5Y R A Y-3'; m5Y (m5C or T), R (A or G), Y (C or T). This consensus sequence displays an unusually high degree of degeneracy. Also, interesting deviations from this consensus sequence, including a one base-pair deletion in the middle, are sometimes observed in high-affinity MDBP sites.
Insights
Methylated DNA-binding protein (MDBP) recognizes specific DNA sequences. While typically requiring DNA methylation for binding, some human MDBP sites bind independently due to thymine substitution.
Area of Science:
- Molecular Biology
- Genetics
- Epigenetics
Background:
- Methylated DNA-binding protein (MDBP) is a mammalian protein that binds to methylated DNA.
- Specific DNA sequences in plasmids and the human genome are recognized by MDBP.
Purpose of the Study:
- To identify and characterize high-affinity MDBP recognition sites in the human genome.
- To investigate MDBP binding to viral DNA sequences and understand the consensus recognition sequence.
Main Methods:
- Cloning of MDBP recognition sites from the human genome based on MDBP binding.
- Identification of MDBP sites in herpes simplex virus type 1 (HSV-1) DNA.
- In vitro methylation assays to assess binding requirements.
Main Results:
- Three high-affinity MDBP sites were cloned from the human genome, showing homology to prokaryotic sites but exhibiting methylation-independent binding.
- Three MDBP sites were identified in HSV-1, with two requiring CpG methylation for binding and located upstream of viral genes.
- A degenerate consensus sequence for MDBP recognition was determined: 5'-R T m5Y R Y Y A m5Y R G m5Y R A Y-3', with observed deviations.
Conclusions:
- MDBP recognition sites exhibit sequence degeneracy and variations, including deletions.
- Human MDBP sites can bind independently of methylation, suggesting alternative recognition mechanisms.
- MDBP binding sites are present in viral genomes, potentially regulating viral gene expression.
