Molecular genetics exam 3 short answer/essay

what does Base Excision Repair (BER) fix?

repairs single base damage that does not significantly distort the DNA helix. ex: deamination of cytosine creates a U-G base pair. or depurination =loss of A or G bases

BER steps

1.glycosylase scans and cuts damaged DNA nucleotide base, leaving behind an AP site. 2. APE1 nicks backbone at AP site removing the baseless nucleotide. 3. AP site is excised leaving behind a gap. 4. DNA polymerase fills gap with correct nucleotide using complementary strand as template. 5.DNA ligase seals nick restoring DNA integrity

What does NER fix?

NER fixes bulky, helix-distorting lesions including: Pyrimidine (thymine)dimers caused by UV light, and Bulky chemical adducts

NER steps

1.Proteins detect distortions in DNA helix (mismatched bases also are detected) 

2. Incision: Endonuclease cleaves on both sides of the damaged base i 

3. Excision: exonuclease or helicase removes DNA between nicks  

4. Synthesis: DNA polymerase synthesizes replacement DNA  

5. DNA ligases seals the final nick 

What is the backup plan steps?

Human DNA Polymerase eta can correctly replicate past a T‑T dimer, inserting A‑A bases. If  Dimerization Occurs Between 5’- CC - 3’ Bases or 5’- TC - 3’ Bases , 3’C deaminates to U, then C mutates to T , C-T TRANSITION is most common mutation in skin cancer. 

what are the two Double stranded break repair mechanisms?

1. homologous recombination.

2. non-homologous end joining

pros of homologous recombination

requires a homologous template(sister chromatid or homologous chromosome). Repairs accurately because the undamaged strand guides synthesis. Occurs in S and G2 phases when sister chromatids are available!

pros of non-homologous end joining

doesnt require a homologous template(break ends are directly ligated without need for homologous template) faster but error-prone as ends may be trimmed or filled in causing small insertions/deletions.  

steps of NHEJ

1.End recognition: Ku70/80 binds to the broken DNA ends  

2. DNA‑PKcs recruited → activates synaptic complex formation 

3. End processing:Artemis trims damaged or incompatible ends 

4.Filling: DNA polymerase fills gaps (enzyme unknown) 

5.Ligation: DNA ligase IV + XRCC4 ligate the ends to rejoin. Repair complete 

Summarize NHEJ process

NHEJ repairs double‑stranded breaks by directly ligating DNA ends without using a template. Ku70/80 recognizes the break, DNA‑PK and Artemis process the ends, and ligase IV/XRCC4 seals them. Because ends are trimmed or filled in, NHEJ is inherently error‑prone. 

Difference between Class I and Class II DNA transposable elements

class II- cut and paste or copy and paste, move as DNA not RNA, found in prokaryotes and eukaryotes, contain transposase gene, terminal inverted repeats (TIR’s) and target site duplications(TSD’s).

Class I- Retrosposons RNA mediated move via RNA intermediate, found in eukaryotes, (DNA-->RNA-->reverse transcription-->DNA-->insertion),include: LTR retrotransposons, non-LTR retrotransposons(LINE’s), SINE’s 

What are Class II processes?

Non-Replicative(cut and paste) transposition:the transposon is excised from its original site and inserted elsewhere. IMPORTANT: NO NEW COPY IS MADE, LEAVES A FOOTPRINT AT THE ORIGINAL SITE 

and Replicative (copy and paste) transposition:the transposon stays at the original site AND  a new copy inserts elsewhere. 

IMPORTANT: produces two copies of the transposon, pseudoreplication fork 

Class II Non-replicative (cut and paste) transposition steps

1. Transposase is transcribed 

2. Transposase binds the terminal inverted repeats--> forms the transpososome(synaptic complex) 

3. Nicking at the ends of the transposon-cuts occur at the TIR’s and the two mechanisms possible for excision are hairpin intermediate or direct double strand cleavage 

4. Excision of the transposon-hairpin ends are opened by transposase if needed 

5. Target site attack-3'OH groups on transposon perform nucleophilic attack on staggered cuts in target DNA  

6. Insertion + target site duplication(TSD)- gaps filled by host polymerases creating duplicated target site repeats 

7. Original site repaired by NHEJ or homologous recombination 

Class II Replicative(copy and paste transposition steps:

1.  the transposase binds TIR’s--> forms transpososome  

2. Nicking occurs but the transposon is not excised 

3. Strand transfer creates ‘pseudoreplication fork’. Transposon becomes connected to both donor and target DNA  

4. Replication machinery copies the transposon 

5. Resolution: site specific recombination may resolve cointegrate structures 

Steps of Class I LTR retrotransposons transposition

Resemble retroviruses but cannot exit the cell. IMPORTANT: have LTR’s at both ends, resemble retrovirus 

Steps: 

1. Transcription from promoter in 5’LTR 

2. Translation -produces Gag and Pol(reverse transcriptase, VLP monomers, integrase) 

3. VLP(virus-like particle) assembly in cytoplasm 

4. Reverse transcription inside VLP- RNA--> cDNA--> second DNA strand synthesized 

5. Nuclear import+ integration- integrase inserts the cDNA into the genome 

Steps of Class I non-LTR Retrotransposons (LINE’s) transposition

Ex. LINE –1(L1)-17% of human genome. IMPORTANT: autonomous(encode their own machinery), no LTR’s, move via TPRT not VLP’s 

Steps: 

1. Transcription from internal promoter 

2. Translation – produces ORF1(RNA-binding protein), ORF2(endonuclease + reverse transcriptase) 

3. RNP formation- LINE RNA + ORF1 + ORF2 assemble in cytoplasm 

4. Target-primed reverse transcription (TPRT)- ORF2 endonuclease nicks genomic DNA, the 3’OH of the nick primes reverse transcription directly at the target site 

5. Second-strand synthesis + integration 

Differences between LTR