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A uniformly labelled (32P) single-stranded DNA (ssDNA) was incubated with a homologous double-stranded DNA (dsDNA) in the presence of Rad51 and/or RPA
Question

A uniformly labelled (32P) single-stranded DNA (ssDNA) was incubated with a homologous double-stranded DNA (dsDNA) in the presence of Rad51 and/or RPA along with ATP or the non-hydrolyzable ATP-γS to study a three-strand exchange reaction. The reactions were terminated at various time points, and DNA was digested with EcoRI followed by electrophoresis and autoradiography. Results are shown in the figure below.

Based on the above data, which one of the following statements is INCORRECT?​

A.

Rad51 requires ATP hydrolysis for strand exchange reaction.

B.

Strand exchange does not take place in the absence of RPA.

C.

The polarity of strand-exchange reaction is in the 3’ to 5’ direction.

D.

The rate of strand-exchange is 7 kb/hour.

Correct option is C

The experiment analyzes the three-strand exchange reaction and the role of Rad51 and RPA proteins, along with ATP or ATP-γS, in the strand-exchange mechanism. The reactions were terminated at various time points, and EcoRI was used to digest the DNA. The resulting DNA fragments were analyzed using electrophoresis and autoradiography.

Key Observations:

  1. The formation of hybrid DNA is observed at different time points, and the extent of the reaction depends on the presence of Rad51 and RPA, as well as ATP.
  2. When ATP-γS (a non-hydrolyzable form of ATP) is used, strand exchange does not occur as efficiently as when ATP is present.
  3. Strand exchange requires RPA, as there is no formation of hybrid DNA without it.
  4. The rate of strand-exchange is approximately 7 kb per hour, based on the observed time-course data.

Analysis of Statements:

  • Statement 1:Rad51 requires ATP hydrolysis for strand exchange reaction.

    • Correct. ATP hydrolysis is essential for Rad51 to facilitate strand exchange, as the reaction does not occur with ATP-γS, a non-hydrolyzable form of ATP.
  • Statement 2:Strand exchange does not take place in the absence of RPA.

    • Correct. The data shows that in the absence of RPA, strand exchange does not occur, as no hybrid DNA is formed.
  • Statement 3:The polarity of strand-exchange reaction is in the 3’ to 5’ direction.

    • Incorrect. The typical polarity for strand exchange reactions facilitated by Rad51 occurs in the 5’ to 3’ direction, not the reverse. This statement contradicts the known mechanism of strand exchange.
  • Statement 4:The rate of strand-exchange is 7 kb/hour.

    • Correct. Based on the experiment, the rate of strand exchange has been calculated to be approximately 7 kb per hour.

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