DNA REPAIR MECHANISMS

Exam 2

What are the mechanisms of proofreading during DNA replication?

DNA polymerase and Exonucleolytic proofreading

How do the mechanisms of proofreading during DNA replication work?

• DNA polymerase: Physically struggles with adding the wrong nucleotide

• Exonucleolytic proofreading: If the incorrect nucleotide is added, DNA polymerase stops, removes it from the strand, and replaces it with the correct nucleotide before continuing on (3’-5’)

What are the steps (and key proteins) of strand-directed mismatch repair systems in bacteria?

• Where the mismatch occurs, the correct base is located on the methylated template strand and the incorrect base occurs on the newly synthesized unmethylated strand (MutL protein)

• Enzymes detect mismatch and make a single strand break in the unmethylated strand (MutS protein)

• Nucleotides are cut out on the unmethylated strand (MutL protein)

• DNA polymerase fills in the gap in the 5’-3’ direction

• DNA ligase links new and old nucleotides

What are the steps (and key proteins) of strand-directed mismatch repair systems in eukaryotes?

• Where the mismatch occurs, the correct base is located on the methylated template strand and the incorrect base occurs on the newly synthesized unmethylated strand (the presence of single-strand gaps in the lagging strand before Okazaki fragments are ligated, and the orientation of sliding clamp proteins)

• Enzymes detect mismatch and make a single strand break in the unmethylated strand (MutS)

• MutS recruits MutL and scans the DNA, when sliding clamp is encountered MutL is activated and initiates strand removal

• DNA polymerase fills in the gap in the 5’-3’ direction

• DNA ligase links new and old nucleotides

What are the mechanisms for the repair of damage on one strand of DNA (what type of damage do they repair)?

• Base excision repair (single nucleotide damage)

• Nucleotide excision repair (larger DNA lesions)

What are the steps of Base excision repair?

• DNA glycosylase (recognizes and removes abnormal or incorrect bases from DNA) recognizes the altered base and catalyzes its hydrolytic removal

• Missing base in DNA is recognized by AP endonuclease (repairs DNA by cutting sugar) which cuts the phosphodiester backbone

• DNA polymerase adds a new nucleotide using the other strand as a template

• DNA ligase seals the break

What is the template for Base excision repair?

The undamaged complementary DNA strand

What are the steps of Nucleotide excision repair?

• DNA lesion is recognized by a large multienzyme complex which scans for discrepancies in the double helix

• The complex breaks the backbone of the abnormal strands on either side of the lesion

• DNA helicase peels away the single strand containing the damage

• Gap is repaired by polymerase and ligase

What is the template for Nucleotide excision repair?

The undamaged complementary DNA strand

What are translesion DNA polymerases (what do they do)?

The chemically reverse DNA damage by replicating DNA through the damage

What are the mechanisms for the repair of double-stranded breaks in DNA?

• Nonhomologous end joining

• Homologous recombination

What are the steps of Non-homologous end joining (NHEJ)?

• Broken ends are processed to remove damaged nucleotides by nuclease

• Trimmed ends are brought together by DNA ligase, which ends up deleting that portion of the DNA sequence as some base pairs are lost at the break site

What is the template for NHEJ?

There is no external template, the broken DNA ends serve as a “guide”
for repair.

What are the steps of Homologous recombination?

• Damaged helix is brought into proximity with a homologous intact DNA helix

• Nuclease digests broken strand ends

• Strand exchange occurs (one of the 3’ overhanging strands searches the template double helix for the homologous sequences by base-pairing

• When stable base-pairing is achieved DNA polymerase extends the invading strand using the undamaged template

• Invading strand is released

• DNA synthesis continues (using remaining segments of damaged strands as templates)

• DNA ligase brings everything together

What is the template for Homologous recombination?

Another DNA double helix which needs to be nearly identical in sequence