Exam 2: Lecture 7 Flashcards Preview

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Flashcards in Exam 2: Lecture 7 Deck (13):
1

Double Stranded Breaks

-one of most toxic forms of DNA damage
-characterized by breakage of phosphodiester bonds that keeps both polynucleotide strands together
-result most commonly from exposure to various forms of radiation as well as physical contratins that are placed on double helix during DNA synthesis, mitosis, and meiosis
-if not repaired cell will most likely undergo apoptosis (programmed cell death)
-DNA damage repair mechanisms that we have already discussed (Mismatch Repair, Base Excision, Nucleotide Excision, Fail-Safe Glycosylase etc) are not used to detect or repair this type of damage
-detected by different mechanisms
-if correctly repaired cell will survive without any change to genome sequence
-if repaired incorrectly leads to sequence changes in genome
-in the cases were these sequence changes inactivate tumor suppressor genes then this will lead to tumor formation (tumorigenesis)

2

Repairing Double Stranded Breaks: Non-Homologous End Joining (NHEJ)

-the double stranded breaks are essentially repaired by directly joining the fragments back together
-set of enzymes will be recruited to the sites of the double stranded break
-these and other proteins will then ligate the ends of the fragments back together
-most often used when a deletion has not been generated – just a break

3

Repairing Double Stranded Breaks: Homologous Recombination

-once ends of the fragments are recognized a nuclease is recruited to digest away a short stretch of DNA (resection)
-this nuclease removes a section of each polynucleotide strand
-once this has occurred the digested DNA is aligned with the homologous region of the homologous chromosome
-repair machinery will use the homolog as a template for DNA polymerase to recreate the missing pieces
-set of enzymes are then used to separate the strands and rejoin the fragments
-most effective when a short stretch of DNA has been deleted and must be recreated

4

DNA Damage Checkpoints

-DSB's must be fixed prior to a cell undergoing mitosis or else there will be segregation defects which can ultimately lead to disease or cell death
-if number of DSBs are particularly high may not be enough time to repair all of breaks during interphase
-if this is the case then the DNA damage checkpoint (ATM and ATR) is engaged and cell is prevented from entering mitosis until the breaks are completely repaired
-repair of double strand breaks most likely causes delays in G1 and G2 phase progression
-if breaks are too numerous or if the breaks are not repaired correctly then the cell is directed towards the cell death pathway
-if DSB checkpoint pathway is also defective then tumorigenesis and cancer can result

5

Single Stranded Break

-induction of single strand breaks (SSBs) is also a trigger for the cell to delay its entry into mitosis
-such breaks are induced by exposure to harsh environmental factors
-also also made during the execution of normal cellular processes such as DNA replication, Mismatch Repair, Base Excision Repair and Nucleotide Excision Repair
-repair of single strand breaks most likely cause delays in S phase progression

6

DNA Damage Checkpoint (Not Engaged)

-if not engaged then defects in DNA replication and chromosome segregation occur at high frequencies
-if DSB occurs within tumor suppressor gene, cell will divide uncontrollably which leads to tumor formation
-increase in cell proliferation that is coupled to chromosome segregation and replication defects can lead to cancer

7

Histones Repair DSBs

-interact with double helix along entire length of chromosome they're well positioned to serve as recruiting scaffold to areas of DSBs
-nucleosome is made up of histone proteins H2A, H2B, H3 and H4
-about 10% of H2A is a special variant H2AX that's encoded by separate gene

8

H2AX

-constitutively phosphorylated under normal cellular conditions by the atypical kinase WSTF at Tyr-142
-within minutes of DSB induction of H2AX Tyr-142 is dephosphorylated by Eyes Absent (EYA) phosphate
-H2AX then re-phosphorylated at Ser-139 by the ataxia telangiectasia mutated (ATM) kinase
-this Ser-139 phosphorylated version of H2AX is now gamma(y)H2AX and now serves to recruit several DNA repair enzymes like MDC1 and MRN complex
-after enzymes are recruited gammaH2AX is dephosphorylated by PP2A and PP4C phosphatases then removed from chromosome
-in absence of EYA protein, H2AX (Ser-139, Tyr-142) recruits cell death machinery instead

9

Eyes Abscent

-first identified in Drosophilia
-gene cloned by Nancy Bonini
-spontaneously induced deletion of an eye specific enhancer element results in the complete loss of the compound retina
-forced expression of eye in non-retinal tissues is sufficient to induced ectopic eye formation (eye can be functional depending on location)

10

EYA Protein

-contains two distinct functional domains
-first is phosphate domain which is used among other things to remove phosphate group fro Tyr-142 of H2AZ
-dephosphorylation of Tyr-142 is required for subsequent phosphorylation of H2AX ast Ser-139
-second is transactivation domain which is used ot activate transcription
-EYA does not bind to DNA so it can't activate transcription of individual genes on its own
-must physically interact with DNA binding protein
-in developing eyes of insects and vertebrates EYA binds to a transcription factor called Sine Oculis

11

EYA as Transcription Activator

-Ilaria Rebay and Graeme Mardon determined that EYA functions as protein tyrosine phosphatase
-Francesca Pignoni and Ilaria Rebay demonstrated that in addition to its role as phosphatase, EYA also functions as transcriptional co-activator
-in metazoans gene expression is controlled spatially and temporally which means that developmentally regulated transcription factors will bind to enhancer elements and will activate or repress gene transcription by interacting with basal transcription factor machinery and RNA polymerase
-some transcription factors contain a DNA binding domain and a transcriptional activation domain
-other situations these two activities are divided amongst two proteins that physically interact (case for Sine Oculis)-Eyes Absent composite transcription factor
-Sine Oculis contains DNA binding domain while Eyes Absent contains transcriptional activation domain
-when two proteins are bound to each other they will bind to enhancer elements within target genes and will activate transcription through direct interactions with RNA polymerase or through interactions with HAT proteins or basal/general transcription factor machinery

12

Vertebrate EYA Proteins

-vertebrate genome contains four copies of eyes absent gene that are called Eya1, Eya2, Eya3 and Eya4
-arose through two sets of gene duplications
demonstranted that mouse Eya1-4 genes can restore eye development to the fruit flies that harbor mutations within the eya gene
-showed that forced expression of Eya1-4 can induce ectopic eye formation in flies
-experiments suggest that the function of the Eya genes have been conserved across 500 million years
-the mammalian genome also harbors 6 sine oculis homologs that are called Six1, Six2, Six3, Six4, Six5, Six6
-rescue and forced expression experiments have been done with the Six1-6 genes. These genes appear to be functionally conserved as well

13

EYE Diseases

-all four Eya genes are expressed in the retina, albeit in different patterns
-some children that are born with cataracts have lesions within the Eya1 gene
-this condition cannot be corrected with surgery and therefore children born with this condition have permanently disrupted vision
-Eya1-4 are including the head, brain, muscle, kidney just to name a few tissues
-human patients that harbor mutations in the Eya1 gene suffer from Branchio-oto-renal (BOR) Syndrome.
-an autosomal dominant disorder that affects the neck, ears and kidney. In extreme cases, an afflicted individual will suffer from complete hearing loss and will be with underdeveloped kidneys – this leads to complete renal failure early in life