Mutations_09.27-09.29

• UvrA + UvrB + UvrC
• three other proteins
• UvrD
• helicase
• DNA Pol I
• DNA ligase

Often they are discovered because of defects in the repair pathways that gives rise to diseases

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder in humans in which the NER pathway is disrupted

XP occurs in both sexes and all races with an incidence rate of 1:250,000

XP patients suffer from extreme UV light sensitivity and are prone to skin cancers (>1000 higher risk!)

DNA pol δ or ε

XPC (assisted by Rad23B or HR23B) recognizes the lesion

XPA binds to the pyrimidine dimer and helps to recruit other proteins to the complex

XPB and XPD (TFIIH subunits) are helicases that separate the DNA strands around the damage

•  
• RPA keeps DNA strands apart

XPF/ERCC1 dimer and XPG are endonucleases that cut the DNA on either side of the damage

The cut fragment is removed and the gap is filled in by DNA Pol δ or ε

• Rad23B or HR23B
• Sensitive to radiation damage, HR23B is the human homolog of this gene

transcribed strands of transcriptionally active genes are fixed first, the non-transcribed strands are repaired next, and the transcriptionally silent DNA gets mended last

• TC-NER (transcription-coupled NER)
• GG-NER (global genome NER)

• EMS
• nitrogen mustard gas

Ionizing Radiation, such as XRay

UV-B radiation

alkylating agents, like EMS, mustard gas

• DNA insertion with agro bacterium in plants (Ti-plasmids)
• Viruses
• Transposons

bulky adducts, UV-radiation

• zinc fingers
• talens
• CRISPR-cas9

• allkylating agents
• oxidizing agents (like hydrogen peroxide)

In TC-NER, RNA polymerase stalls when it encounters distorted DNA and triggers assembly of the repair machinery

In GG-NER, DNA lesions are detected by specialized damage sensing proteins, including XPC-Rad23B and DNA-damage binding (DDB) complexes that constantly scan the genome and recognize helix distortions

TC-NER pathway being triggered

RNA Pol stalls when it runs into damage in the template strand

CSA and CSB (proteins deficient in the patients with Cockayne's syndrome!) help to recruit TFIIH to the damaged site and, possibly, to displace RNA pol

XPB and XPD helicase subunits of TFIIH unwind a 20-30 nucleotide stretch of DNA (including the damaged region) in an ATP-dependent manner

XPA, XPG and RPA are then recruited

in patients with Cockayne's syndrome

• the XPB and XPD helicase subunits of TFIIH

• XPA
• XPG
• RPA

Common pathway shared by TC-NER and GG-NER following damage detection

RPA keeps DNA strands apart

XPF/ERCC1 heterodimer binds next

XPG (related to FEN-1 in BER!) cuts 2-8 nt downstream of the lesion, while XPF/ERCC1 cuts 15-24 nt upstream of the lesion

Next, replicative gap-repair proteins RFC, PCNA, and DNA Pol δ or ε bind to the 3'-OH group (arrowhead) generated by the XPF/ERCC1 cut and carry out new DNA synthesis that fills the gap

This leads to displacement of the damage-containing oligonucleotide and of TFIIH, XPA, XPG, and XPF/ERCC1

The final nick is sealed by DNA ligase I

FEN-1

• Recessive autosomal disorder caused by mutations in the genes of TC-NER
• Characterized by:
• light sensitivity in some cases
• neurological abnormalities
• premature aging of some tissues
• facial and limb abnormalities
• Dwarfism
• early death due to neurodegeneration

the photosensitive form of this disease results from mutations in the subunits of the TFIIH complex involved in NER

MMR corrects mismatches (wrongfully incorporated nt or small insertions/deletions) immediately after DNA replication, increasing replication fidelity by 102-103 fold

Mismatch repair systems scan newly-replicated DNA duplexes for mismatched bases, i.e., for distortions in the double helix

In E. coli MMR machinery identifies the parental strand based on its full methylation status, removes mismatched base(s) on the unmethylated strand and replaces them using methylated DNA as a template

Mismatch repair (MMR)

MutS, MutL, and MutH are essential for detecting the mismatch and recruiting repair machinery

MutS dimer recognizes and binds the mismatch

MutH endonuclease binds to hemimethylated GATC sites

MutL brings the two together forcing the DNA to loop out

MutH gets activated by MutL and nicks the newly synthesized strand next to GATC

UvrD helicase

• False
• The detailed mechanism for MMR is unclear

• MMR
• MutL=hMutL(MLH)
• MutS=hMutS(MSH)

Defects in either MLH or MSH result in enhanced susceptibility to cancer (i.e., HNPCC = hereditary non-polyposis colon cancer, aka Lynch syndrome)

• False
• At least not for all of them
• MutL=hMutL(MLH)
• MutS=hMutS(MSH)
• MutH=N/A

it deals with recognizing GTC, which isn't a problem in eukaryotes

by recognition of hte mismatch or insertion/deletion loops (IDL)

• The predominant base-base mismatch and single-base IDL recognition activity in human cells is provided by MSH2-MSH6

• base-base mismatch
• single-base IDL recognition

• Recognize larger insertion/deletion loops (IDLs)

DNA Pol IV, Pol V both are involved in repair mechanism of translesion synthesis.

DNA Polymerase II 3′ – 5′ exonuclease activity restart replication after replication stops due to DNA strand damages.

• TdT is a non-template directed DNA polymerase dT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity.
• Increases the variety of antigen receptors that a cell is equipped with to fight pathogens.

• Polymerase γ is a Type A polymerase, whose main function is to replicate
• and repair mitochondrial DNA. It also functions by proofreading 3′ to
• 5′ exonuclease activity. Mutations on Poly γ significantly affect the
• mitochondrial DNA causing autosomal mitochondrial disorders.

Pol δ and ε also have a 3′ to 5′ exonuclease activity.

• Pol α works by binding to the primase enzyme, forming a complex, where they both play a role in initiating replication. Primase enzyme creates and places a short RNA primer which allows Pol α to start the replication process.
• Pol δ starts the synthesis of the lagging strand from Pol α, while Pol ε is believed to synthesize the leading strand during replication.
• Studies indicate that Pol δ replicates both the lagging and leading strand. Pol δ and ε also have a 3′ to 5′ exonuclease activity.

• Pol β has a short-patch base excision repair mechanism where it repairs alkylated or oxidized bases.
• Pol λ and Pol μ are important for rejoining DNA double-strand breaks due to hydrogen peroxide and ionizing radiation, respectively.