BICD 158 Problem Set 3

BRCA1 and 53BP1 determine the choice of what two major DNA repair pathways?

Explain briefly what each of these two proteins does.

The two major DNA repair pathways that BRCA1 and 53BP1 are involved with are Homology Directed Repair (HDR) and Non-Homologous End Joining (NHEJ). BRCA1 plays a role in promoting DNA repair using homology directed repair. 53BP1 plays a role in promoting non-homologous end joining as a DNA repair mechanism via Ku70 and Ku80.

NHEJ

Non-homologous end joining (NHEJ) is a DNA repair mechanism in which a double-stranded break in DNA can be repaired using proteins called Ku70 and Ku80. These proteins stick to the broken ends of DNA and bring them together such that a ligase protein is able to join the ends together. If the break in DNA is a clean break, then NHEJ is generally an accurate pathway.

MMEJ

Microhomology-mediated end joining (MMEJ) is another method of DNA repair when a double-stranded break is made in the DNA. In MMEJ, small repeat sequences are used to join the ends of the break in the DNA, but as a result, there can likely be deletions that can occur during this process. This could result in framing issues of this sequence, making this not the preferred method for DNA repair.

SDSA

SSA is a DNA repair method that takes information from an intact, uncut chromosome that is homologous to the broken chromosome and uses this information to repair this DNA break. This process leaves the uncut chromosome completely intact

HDR

Homology directed repair (HDR) is a DNA repair process in which a double-stranded break in DNA is repaired by copying the genetic material from the homologous chromosome. To do this, one of the strands of DNA that was broken is resected such that there is a 3' overhang that can interact with either a sister chromatid or homologous chromosome, by making a D-loop, and copy the information from the intact chromosome. Then the repaired strand will return to normal.

Indicate when during the cell cycle that they (DNA repair pathways) act as a primary repair pathway

In the cell cycle, NHEJ acts as a primary repair pathway the whole time. MMEJ is a primary repair pathway during the M phase of mitosis, specifically right before anaphase occurs. However, MMEJ can also sometimes act during the S and G2 phases as well. HDR is a primary repair pathway during the S and G2 phases. And SSA is a primary repair pathway that is used in the S-phase, G2 phase, and into the M phase.

Briefly describe the difference between mitosis and meiosis. Be sure to mention in what cell types these cell division pathways take place and what the final outcome of those divisions are in terms of the numbers of chromosome sets (1 set = 1n). During which of these cell division processes does crossing-over take place? In simple terms, what does crossing over accomplish?

The difference between mitosis and meiosis is that mitosis occurs in somatic cells and meiosis occurs in germline cells. Mitosis is a process in which two daughter cells are produced that are identical to the parent cell. Whereas meiosis produces four daughter cells that are not identical to the parent cell. The daughter cells have a ploidy of 2n (diploid), whereas the ploidy of the daughter cells of meiosis is just n (haploid). Crossing-over occurs during meiosis and not mitosis, which allows for recombination of the parental chromosomes to occur, which is what makes the daughter cells not identical to the parent cell.

The Synthesis-Dependent Strand Annealing (SDSA) branch of the homology dependent repair (HDR) pathway is largely responsible for directional gene-conversion events. What is the key feature of this repair process? What is the primary mechanism that determines the size of the copied region from repair template chromosome?

When repairing a DNA break using the SDSA branch of HDR, the broken strand is retracted such that there is a 3' overhang, and RAD51 binds to the end which allows for the formation of a D-loop. The key feature of this repair process using SDSA is that information from the sister chromatid or homologous chromosome in order to copy the genetic information such that it can repair the break. The primary mechanism used to determine the size of the copied region is how much the strand is resected. ??

From what branch of life did CRISPR/Cas systems originate? What is its purpose in those organisms?

CRISPR/Cas9 systems originated from the bacterial branch, specifically in dairy bacteria. In these dairy bacteria, CRISPR/Cas9 as an immunity system that protects itself from phages and other harmful things that could damage the bacterial genetic material.

What two components from the native Cas9 (Type II) CRISPR system were joined together to create the widely used synthetic two-component system? What are the two synthetic components of this Type II CRISPR system called?

The two compounds that were joined together to make the widely-used synthetic two-component system are crRNA and tracrRNA. These two components are referred to as the sgRNA when put together. This way, all that is needed to make a specific cut in the DNA is the sgRNA and the Cas9 protein.

Cas9 has two active catalytic centers (RuvC and HNH domains). What enzymatic activity is manifested by mutants lacking either or both of these enzymatic functions?

In Cas9, RuvC is a nickase that cuts the strand of DNA that is not hybridized to the RNA and HNH is a nickase that cuts the DNA strand (and RNA) that is hybridized to the RNA. So if Cas9 has mutant RuvC, only the hybridized strand of DNA will be cut. Similarly, if the Cas9 is mutant for HNH, then only the non-hybridized strand of DNA will be cut. If the Cas9 is mutant for both RuvC and HNH then the DNA will not be cut at all.

For typical gene editing of target genes in cultured mammalian cells what additional reagent is needed beyond the core CRISPR constituents? What forms can this component take? What DNA repair pathway is typically responsible for generating targeted gene edits?

In order to make targeted gene edits, exogenous DNA repair templates may needed to be added as a reagent. These templates can take the forms of linear double-stranded DNA, linear single-stranded DNA, or circular double-stranded DNA. Homology directed repair is typically the repair pathway used in order to generate the targeted gene edits.

Some bacteriophage produce anti-CRISPR (Acr) proteins to escape Cas9-mediated destruction. Give two examples of mechanisms by which Acr proteins can inhibit Cas9 activity.

Two examples of how Acr proteins can inhibit Cas9 activity includes (1) AcrllC2 can inhibit the crRNA loading to Cas9, therefore inhibiting Cas9 activity and (2) AcrllA4 and AcrllC3 can inhibit crRNA + Cas9 binding DNA, meaning that it cannot make its cut, thus preventing Cas9's function.

Cas9 bound to a gRNA can cleave DNA in an iso-energetic fashion (i.e., the reaction does not require ATP as an energy source). Briefly explain where the energy for DNA cleavage comes from?

The energy for DNA cleavage by Cas9 comes from the energy that is stored in the Cas9 protein when the kink forms in the DNA and RNA is able to get in between the DNA strands. This stored energy can then be used to cleave the DNA.