Scribd
Upload
16 views

Discussion Week 6

biochemistry

Uploaded by

japanesecurrency123
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
16 views

Discussion Week 6

biochemistry

Uploaded by

japanesecurrency123
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 4

Discussion Worksheet – Fall 2023 - Week 6 (DNA Repair)

Group 1. Biochemical studies have shown that amino groups of


bases, such as the N6 of A can react with proteins bound in the
major groove and form crosslinks like the one shown on the right
(N6dA peptide cross-link = N6X).

Primer extension experiments are performed using DNA


Polymerase I or III (Pol.I or Pol.III) using a 5’-end radiolabeled
primer and two different templates.
Template N contains a normal dA located three nucleotides after
the end of the duplex with the primer. Template N6X contains a
N6dA peptide cross-link three nucleotide after the end of the duplex
with the primer, as shown on the right. The length of the single
stranded region of the template is 17 nt.

A - Describe the results obtained for DNA polymerase III and DNA
polymerase I with the normal template (N) and the template containing N6X.

B- Based on these results, propose a function for DNA polymerase I in the process of
replication that has not been covered in class. What protein in eukaryotic cells might
fulfill a similar function?

Primer+17nt

Unextended Primer-
Group 2

Scientists have purified an enzyme from an archae. They incubate this protein with the
substrate shown below, in the absence of any other nucleotides or nucleic acids.

The red
star indicates a radioactive label. They use 4 different versions of this substrate, which
differ by the identity of the nucleotide at position X, as shown on the top of the gel.

After incubation with the enzyme (+), or


without (-), the nucleic acids are fractionated
according to their size and visualized by
autoradiography such that only the
radioactively labeled DNA fragments are
visible. Numbers indicated on the left side
indicate the sizes of DNA molecules; the DNA
products shown on the left ranging from 22-26
nt is a size ladder to help determine the size of
products obtained after incubation with the
enzyme.
A – Based on the nature of the products
obtained after incubation with the enzyme and
how this enzyme acts on the different
substrates, explain below what type of biochemical activity is catalyzed by this enzyme,
and what its substrate preferences are.

B- Explain below in what general DNA metabolism reaction/pathway this enzyme is


expected to function; compare its activity to that of the enzymes known to function in the
equivalent pathway in bacteria, and explain how it is similar and/or different.
Group3.

The structure of an enzyme that binds to DNA is shown below. The right side
shows details of the interaction with the DNA near the active site of the enzyme.

A- Based on the picture shown on the left, what is the major consequence of the
binding of this protein onto DNA?

B – Based on the picture shown on the right, what is the major difference that this
DNA exhibits compared to a normal DNA? )

C– Predict the type of enzymatic activity that the protein would possess if the
DNA bound to the enzyme in this structure corresponded to the enzyme’s
substrate. In which pathway would this activity be required?

D– Predict the type of enzymatic activity that the protein would possess if the
DNA bound to the enzyme corresponded to the product of the reaction
catalyzed by the enzyme. In which pathway would this activity be required?
Group4

Researchers study the function of residue R154 in OGG1. The interaction of this residue
with a DNA containing an 8-oxoG residue is shown at the bottom of the page. Purified
normal OGG1 (WT) and the R154H mutant OGG1 (R154H) are incubated with a radio-
labeled DNA containing an oxoG (oG) residue paired opposite to either a C or an A in
the complementary strand. The radiolabel (*) is in the strand containing the oxoG as
shown below:

*-5’-20nt-8oxoG-15nt-3’
3’-20nt--C/A--15nt-5’

The reaction products are fractionated on a gel and


the result is shown on the right. b-cleavage is a 20
nucleotide long DNA fragment.
A – Explain what biochemical activity or activities of
the OGG1 enzyme result in the production of the b-cleavage product when the WT
enzyme is incubated with the oG/C product.

B – Compare the biochemical activities detected for the WT and R154H OGG1 enzymes
when they are incubated with the different substrates.

C - Predict the genetic consequences of the R154H mutation of hOGG1 if this mutant
enzyme is introduced in human cells. Be specific in your answer and as to what type of
mutation events could be introduced or minimized.

R154

You might also like