Week 4 Notes PDF - DNA Replication and Repair

2/3 ​ DNA replication of Eukaryotic cells- DNA poly 1 and 3 ​ Will not expect to know that DNA poly 1 and 3 is for e coli, but rather to know that DNA polymerase reads the template 3’ to 5’ ○​ Adds the template 5’ to ‘3 ​ In order for nucleases to add new nucleotides to that end, they nee...

2/3 ​ DNA replication of Eukaryotic cells- DNA poly 1 and 3 ​ Will not expect to know that DNA poly 1 and 3 is for e coli, but rather to know that DNA polymerase reads the template 3’ to 5’ ○​ Adds the template 5’ to ‘3 ​ In order for nucleases to add new nucleotides to that end, they need a 3’ hydroxy group ○​ In order to replicate new DNA, you need a template, DNTps, enzyme, and primer ​ The primer is added by a primase ​ Purpose of telomeres is to solve “end-replication problem” ​ RNA primers need to get removed by enzymes, which leaves a gap which is a problem at the end of the chromosomes ○​ This issue isn’t in organisms like bacteria that have circular DNA ​ Single stranded DNA gap elicits a response in cells, making them unhappy ​ DNA replication has to happen on both the strands, both having different polarity ○​ Polarity is what introduces the problem ​ Formation of telomeres ​ Function to protect chromosomes’ end ​ Telomeric shortening contributes to aging ​ Human telomeres consist of 5’ TTAAGGG 3’ ​ G rich srand strand: strand that extends 5’ to 3’ towards the chromosome’s end and terminates in a small single stranded region named the G-rich overhang ​ Length of telomere sequences on chromosomes are different ​ Cells do not like having single-stranded DNA ​ Telomeres, in order to compensate for the problem, they form the t-loop structures. ○​ G-rich overhang (repititive ○​ sequence) will loop over and form base pairs between g-overhang and double-stranded region ​ D-loop: displacement loop: non-sisters exchange genetic material ​ Proteins are bind to the regions are he Shelterin complex ○​ Those that bind to the end ○​ Those that bind to the double-stranded DNA itself ○​ Telomere-associated proteins (those that don’t bind DNA) ​ What happens is that we have this gap that can’t be filled by nucleotides ​ Telomerase extends that G-overhang ○​ By doing this, the other end can get filled in ​ RNP- RNA & Protein ○​ 2 parts that are important to it’s activity are TERT and TERC ○​ TERT- telomerase reverse transcriptase ○​ TERC- non-coding template ​ Telomerase continues to add 6-nucleotide sequence to the end of the chromosomes ​ Cells that aren’t continually dividing stabilize their tips with t-loops ​ If the cell is not continuing to divide–it’s fine because it can form it’s t-loops ​ If you’re in a cell like stem cells that’s continuing to divide, telomerase activity is VERY IMPORTANT b/c everytime that the cell divides the ends will get shorter and shorter ​ Idea of synesis- telomeres become short–cells lose their function ​ Mutation in a gene that regulates the shelterin complex– important for recruiting telomerase to extend tips 2/5 ​ Think about the scale and source of the damage ​ Point mutations are smaller sized mutations ○​ Contrast with larger sized damage ​ Cells have proofreading activity ○​ Sometimes not 100% ​ Thinking about phenotypic consequences– think about where the point mutation is located ○​ A lot of our DNA has non-coding region ​ Point mutations can be substitution, deletions, or insertion ​ Substitution ○​ Purine to pyrimidine is a transversion ○​ A to G or C to T is a transition (purine to purine or pyrimidine to pyrimidine) ​ Different types of damages ​ These 4 nucleotides can experience oxidation, hydrolysis, and methylation, changing their substituents ​ Some of the byproducts of metabolism are reactive oxygen species ​ Deupruination- The nucleotide loses its nitrogenous base ​ Deamination- Removal of an amino group- can change a C to U ○​ If this isn’t fixed before replication, you have gone from a CG to a TA ​ Double-stranded breaks, one of the most deleterious ○​ Caused by ionization radiation ○​ Mechanism that is used to repair this involves homologous recombination ​ Single stranded DNA is easier to repair ​ UV light is bad for DNA because instead of the thymine’s doing a watson crick base pair, 2 thymines come together to make thymine dimers ​ Oxidation of guanine= 8-oxodG mispairs with A ○​ If this pairs with cell that’s dividing a normal G-C pair becomes mutant T-A ​ DNA polymerase itself is really good at picking the complementary nucleotides on the opposite strand ○​ This process has high fidelity ​ If wrong nucleotide is inserted, DNA polymerase will stop ​ 3’ to 5’ exonuclease recognizes and excises mismatches ​ Mismatch repair pathway is active in cells, plays outs its main role in cells that are dividing ○​ Main goal is to fix vast majority of replication error ○​ Recruits accessory proteins, stimulated to cut strand and mismatch ○​ New replication by DNA polymerase, nick sealed by DNA ligase ○​ The signal that actually directs the mismatch complex to the newly made strand disappears after replication ​ PCNA is the clamp ○​ Based on the location of where this is, you can distinguish where the problem is ​ Some other types of DNA damage ​ Repair mechanism that can be found in cells that are differentiated ​ Base is now a Rracil because it’s been deaminated ○​ How is this change monitored? ​ DNA glycosylases can recognize a specific type of altered base in DNA and recruit and endonuclease (endo = nuclease cleaves within polypeptide chain) cuts the phosphodiester backbone (covalent bond); cuts the DNA at specific point ​ Exonuclease cleaves nucleotides one by one ​ DNA ligase repairs ​ Repairs larger damage ​ What you don’t want is for the mutation to be passed to the next cell division ​ P53 is a tumor suppressor and transcription factor - controls expression ○​ Tells cells to stop and repair ​ Normal cells have low levels ○​ Continuously made, transcribed, made into protein, and degraded ​ Mdm2 complex does this ​ DNA damage leads to phosphorylation of p53 -> dissociates from protein -> now is at high levels ○​ How it puts the brakes on is that it induces p21 (CDK inhibitor) which inhibits cyclins ​ Therefore cell can’t pass G1/S checkpoint and can’t enter S phase ​ Normally kept low ○​ In response to dna damage, the kinases atm and atr are activated and phosphorylate p53 ○​ Now p53 is high in response to double stranded break ○​ P53 is a transcription factor that binds many genes 2/7 ​ Cell is deciding it’s division pattern ​ P53 induced in response to cel stresses like single or double stranded breaks ​ DNA damage can be exogenous- something from environment ○​ Can also be endogenous- errors from polymerase, etc ​ Cancer is not due to a single mutation- rather it’s multiple mutations over time ​ DNA polymerase sometimes slips up ​ Defects in polymerase can lead to expansion ​ Huntington disease is caused by expansion ​ Interstrand cross links ​ Nucleotides are held together by covalent bonds ○​ Bases are held by hydrogen bonds ​ Point mutations happen on one of the 2 strands- involve insertions or deletions ○​ Consider the type or scale ​ This is a very large type of DNa damage ​ Chromosomal level damages ​ Aneuploidy- less or more chromosomes than normal ​ Trisomys arise because of defects in meiosis ​ ​ In rearrangements can occur after double stranded breaks ○​ Regions can get deleted or inverted in the process of fixing ○​ Depending on how many breaks you have, you can have many different types of rearrangements ​ Spo11 during meiosis induces double stranded breaks ​ When trying to repair double stranded breaks, a lot of times those new fusion of chromosome parts creates new genes ​ NHEJ- is two ends coming together and fusing ○​ Not a great way to repair because nucleotides can get lost ○​ Used predominately G0, G1, and S phase ○​ VERY error prone- causes mutations ​ Homologous directed repair AKA homologous recombination ○​ Less error prone ○​ MUST have a template- which is why it only happens during S phase and G2 ​ Because we know in S phase we just replicated a sister chromatid, so if a break happens in one, we have another ​ Can use sister chromatid (identical) -> error free ​ Homologous chromosome (similar)-> may introduce minor differences (occurs when a sister is not available)