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Biochemistry

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Testing a Transcription-Related Hypothesis

BIOCHM-A77KTL

DNA repair mechanisms are necessary for preserving the integrity of the genome. However, mutations can sometimes be advantageous. Activated B cells upregulate expression of activation induced deaminase (AID), which is recruited to genes encoding the B cell receptor (BCR) and deaminates cytosines into uracils on single-stranded DNA.

When AID acts on cytosines in switch regions of the heavy chain (IgH) genes, double-stranded breaks (DSBs) can occur in these regions during the process of DNA repair and the IgH locus undergoes a process called class-switch recombination (CSR).

In addition to AID expression, CSR requires active transcription in order to provide AID with a substrate.

Researchers have observed significantly reduced CSR in the absence of Parp3, a DNA damage repair protein. and want to test whether this is due to aberrant transcription of or transcriptional stalling at the IgH switch regions.

Which of the following experiments best evaluates these hypotheses?

A

Measure switch region transcripts at various time points post-activation of wild-type and Parp3-/- B cells using qPCR.

B

Perform a yeast two-hybrid screen using RNA polymerase II as the protein and one of the switch regions as a DNA substrate.

C

Measure switch region transcripts at various time points post-activation of wild-type and Parp3-/- B cells using qPCR and perform ChIP-qPCR at the switch regions.

D

Perform ChIP-qPCR at the switch regions.