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Potential involvement of t-circles in the Alternative Lengthening of Telomeres in Naumovozyma castellii

Kopczyk, Aleksandra (2024) MOBN03 20232
Degree Projects in Molecular Biology
Popular Abstract
Merry Go Round: Rolling-circle telomere elongation

Imagine a shoelace. At the end of the shoelace, there is a protective piece called an aglet, keeping the fibers together. A human chromosome is a bit like that, too – long and linear, with a characteristic structure at the end called the telomere, which protects the chromosome from instability.

With each cell division, a piece of the telomeric DNA is lost. When telomeres become critically short, cells stop dividing and eventually die. Most cells counteract this telomere shortening by expressing the enzyme telomerase. In the absence of telomerase, however, alternative ways of telomere elongation might be activated, collectively termed as Alternative Lengthening of Telomeres (ALT)... (More)
Merry Go Round: Rolling-circle telomere elongation

Imagine a shoelace. At the end of the shoelace, there is a protective piece called an aglet, keeping the fibers together. A human chromosome is a bit like that, too – long and linear, with a characteristic structure at the end called the telomere, which protects the chromosome from instability.

With each cell division, a piece of the telomeric DNA is lost. When telomeres become critically short, cells stop dividing and eventually die. Most cells counteract this telomere shortening by expressing the enzyme telomerase. In the absence of telomerase, however, alternative ways of telomere elongation might be activated, collectively termed as Alternative Lengthening of Telomeres (ALT) mechanisms. It has been suggested that circular DNA molecules containing the telomeric sequence (a t-circle) may be involved in the ALT mechanism. Such structures have been observed in a variety of organisms, including human cancer cells, yeast, plants and amphibians. The “roll and spread” model suggests that the telomeres might be elongated by the rolling-circle replication, using the t-circle as a template.

Detecting t-circles in budding yeast Naumovozyma castellii
Budding yeast is an exceptionally useful model organism to study many molecular processes. It is simple in handling, while exhibiting many similarities to human cells. In our lab, we use the budding yeast Naumovozyma castellii as a model organism to study telomere biology. It easily switches to using the ALT mechanism of telomere elongation upon inactivation of telomerase. Thus, we wanted to investigate whether ALT N. castellii cells contain t-circles that could be potentially involved in this mechanism.

To investigate whether t-circles are present in ALT N. castellii cells, we adapted the Rolling Circle Amplification (RCA) assay. Establishment and optimization of this method was a major part of this project. The RCA assay is based on the activity of phi29 DNA polymerase, which can use self-primed t-circles to generate a long single-stranded product that is easy to detect through blotting and hybridization with an appropriate probe. We introduced several improvements to the assay, including the use of a more efficient phi29 DNA polymerase and more precise blotting technique.

We obtained many generations of telomerase-deficient N. castellii cells and confirmed their ALT phenotype by evaluating their telomere structure. We have subsequently tested them for the presence of t-circles using the RCA assay. Based on our preliminary results, it seems that ALT N. castellii cells might contain t-circles. The RCA signal does not appear to be consistent throughout different generations and varies between different strains, which might reflect the dynamic nature of the ALT mechanism.

Master’s Degree Project in Molecular Biology 60 credits 2024
Department of Biology, Lund University

Advisor: Dr. Marita Cohn
Advisor’s Department: Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Kopczyk, Aleksandra
supervisor
organization
course
MOBN03 20232
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9176959
date added to LUP
2024-10-23 11:24:22
date last changed
2024-10-23 11:24:22
@misc{9176959,
  author       = {{Kopczyk, Aleksandra}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Potential involvement of t-circles in the Alternative Lengthening of Telomeres in Naumovozyma castellii}},
  year         = {{2024}},
}