Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Area of Science:

• Plant molecular biology
• Genetics
• Transposable elements

Background:

• Activator (Ac) transposition in maize is thought to be linked to DNA replication.
• The non-autonomous Dissociation (Ds) element is a derivative of Ac.
• Previous studies suggest a correlation between Ac/Ds excision and replication timing.

Purpose of the Study:

• To directly investigate the role of extrachromosomal DNA replication in Ds excision.
• To compare Ds excision frequencies from replicating and nonreplicating vector DNAs.
• To provide direct evidence for replication-dependent transposition in maize.

Main Methods:

• Transfection of black mexican sweet maize protoplasts with engineered vector DNAs.
• Construction of replicating (rep+) and nonreplicating (rep-) extrachromosomal vectors containing a Ds element.
• Co-transfection with a transposase expression vector to facilitate Ds excision.
• Quantitative reconstruction experiments to determine excision frequencies.

Main Results:

• Ds excision was detected only in cells transfected with replicating Ds-vector DNA and transposase.
• Ds excised at a frequency at least 3 x 10^5-fold higher from replicating vector DNA compared to nonreplicating vector DNA.
• These findings demonstrate a strong coupling between Ds excision and vector DNA replication.

Conclusions:

• Extrachromosomal vector DNA replication is a critical factor promoting Ds excision in maize.
• The results provide direct experimental evidence for replication-dependent transposition of the Ds element.
• This study clarifies the mechanistic relationship between DNA replication and transposable element activity.