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A 189-bp repeat region within the human cytomegalovirus replication origin contains a sequence dispensable but
1The Institute of Medical Science, The University of Tokyo, Tokyo, 108, Japan.
Insights
The human cytomegalovirus (HCMV) replication origin requires a specific internal sequence within its 189-bp repeat region for DNA synthesis. Modifications to this sequence abolish replication, but copy number changes do not affect it.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Human cytomegalovirus (HCMV) replication origins show strain-dependent variations in the copy number of a 189-bp repeat region.
- Previous studies indicated that a near-complete deletion of this region did not abolish origin function.
Purpose of the Study:
- To investigate the functional significance of the 189-bp repeat region within the HCMV replication origin.
- To determine if specific sequences within the 189-bp region are essential for HCMV DNA replication.
Main Methods:
- Mutagenesis of the 189-bp repeat region in the HCMV replication origin.
- Replacement of internal sequences with lambda DNA of varying lengths.
- Introduction of nucleotide substitutions within the 152-bp internal sequence.
- Assessing replication ability in infected cells.
Main Results:
- Replacement of the internal 152-bp sequence or introduction of nucleotide substitutions abolished replication ability.
- Variations in copy number (one or two copies) of the 189-bp region did not affect replication.
- Inversion of the 152-bp internal sequence maintained wild-type replication ability.
Conclusions:
- The 189-bp repeat region is not merely a spacer but contains an essential, irreplaceable sequence crucial for HCMV DNA replication.
- This irreplaceable sequence likely plays a supporting role in the initiation or maintenance of HCMV DNA synthesis.
Abstract:
The human cytomegalovirus (HCMV) replication origin exhibits a strain-dependent difference in the number of copies of a 189-bp region: the AD169 and Towne strains contain one and three copies of the region, respectively. A nearly complete deletion of the 189-bp repeat region of the Towne strain does not eliminate the origin's ability to initiate DNA synthesis. Here we report that the replication ability of the HCMV replication origin in infected cells disappeared after replacements of an internal sequence (152 bp) of the 189-bp repeat region with lambda DNA of identical and different lengths as well as after introduction of multiple nucleotide substitutions within the 152-bp internal sequence of the 189-bp repeat. In contrast, a variation in the copy number of 189-bp region (either one or two copies) or an inversion of the 152-bp internal sequence of the 189-bp repeat maintained replication abilities similar to those of the wild-type origin of the Towne strain. These results indicate that the 189-bp repeat region within the HCMV replication origin is not just a dispensable spacer sequence but instead contains an irreplaceable sequence that may play a supporting role in HCMV DNA replication.