Notes from the Lab: How Incorrect Repair of Double-Strand DNA Breaks Lead to Cancer
"Notes from the Lab" spotlights innovative work from Columbia investigators, post-docs, fellows, and students addressing problems in cancer research and care.
The Symington Lab
We study the mechanisms of DNA repair, specifically DNA double-strand breaks, using the yeast model system. Double-stranded breaks generally occur due to errors during DNA replication, but they can also be caused by environmental or cellular factors.
The Research
“Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism,” published in Nature Communications.
The Cancer Problem We Are Solving
If it doesn’t kill the cell, erroneous repair of double-strand breaks can lead to what are called gross chromosomal rearrangements. Minor mutations occur at the single nucleotide level, such as base pair substitutions or deletions. But gross chromosomal rearrangements involve duplications, deletions, or translocations on the order of hundreds or thousands of base pairs. For example, a large chunk of DNA from one chromosome will end up going into another chromosome.
Such a phenomenon can cause normal cells to lose a tumor suppressor gene or gain extra copies or activity of an oncogene, increasing the likelihood that they will become cancer cells.
A Bit of Background
A specific type of gross chromosomal rearrangement known as inverted duplication is commonly observed in cancers. In this case, after a break in the chromosome, one part of the chromosome is lost while the other part is duplicated in an inverted manner.
The mechanisms by which inverted duplications arise isn’t fully clear, although they are thought to originate from double-strand breaks. We wanted to investigate the specific effect of the double-strand break on the frequency and the mechanism of gross chromosomal rearrangements. We used CRISPR-Cas9 to induce double-strand breaks in the yeast genome and analyzed the cells that survived the break using molecular techniques such as polymerase chain reaction (PCR) and whole genome sequencing.
What this New Research Uncovers
We found that, if you induce a double-strand break near short naturally occurring inverted repeat sequences — sequences where one half is the reverse complement of the other half — it can prime the formation of large inverted duplications. It happens at a relatively high frequency in cells that lack Sae2, a protein involved in DNA end resection, as well as Mre11, a protein involved in the repair of double-strand breaks.
Essentially, an inverted repeat sequence that becomes single-stranded will fold back on itself, creating a hairpin-capped chromosome. During DNA replication, the sequence will be read going into the hairpin and looping back, thus resulting in a large inverted duplication.
The experiments also uncovered the role of DNA polymerase δ, an essential enzyme normally involved in the synthesis of the lagging strand during DNA duplication, in promoting inverted duplications.
Next Steps
All of this work has been done in yeast, so it would be interesting to see what happens if we induce double-strand breaks near inverted repeat sequences in mammalian cells. Another research direction would be to leverage the availability of cancer genomes in databases. For instance, we could look for signatures of inverted duplications to see if they correlate with mutations in MRE11 or CtIP (Sae2 ortholog). Since inverted duplications are associated with cancers with poor prognosis, inhibiting their formation could have therapeutic potential.
