radiobiology III Flashcards
what is microbeam targeting of cytoplasm?
-dye dyes cytoplasm
-chromosome 11 is dyed in nature
tested three different scenarios
-radiated nucleus (less mutations)
-radiated cytoplasm (more mutations)
-spontaneous mutations
what does cytoplasmic irradiation lead to?
mutations resembling spontaneous mutations via ROS mediated DNA damage which peaks within 5 minutes
-counted surviving mutations
-B50= cause oxidative stress (glutathione depletes so cannot help with ROS)
what is an essential process?
-DNA replication (S-phase) is an essential process in life forms for faithfully transmitting genetic information encoded within the nuclear and mitochondrial DNA to daughter cells curing somatic division and to gametes for the inheritance of “the chemistry of life” to the next generation
what is DNA replication?
-all the DNA molecules on separate chromosomes must be replicated only once per cell division cycle and then evenly segregated during mitosis
-coordinated initiation of DNA replication from multiple origins in each chromosome so that the genome id duplicated in a timely manner during the cell division cycle
-DNA replication is finished by replication termination, which involves the convergence of replication forks, disassembly of the replisome and resolution of the daughter DNA molecules
what is a replisome?
-a multi-protein machinery containing the replicative DNA helicase and polymerases, unwinds template DNA and synthesizes nascent DNA on both leading and lagging strands generating DNA replication forks
what are the tracing origins and direction of replication?
what are the difference in replication timing?
-bi-directional and temporally regulated in large domains (near megabased-sized domains in mammals)
-influenced by different mechanisms, such as rate-limiting DNA replication factors, transcription, epigenetic control, chromosome structure, and 3D chromatin organization in the nucleus
-dynamic and developmentally controlled
is DNA passive or active?
ACTIVE
-ATP dependent
what is the first steps and initiation of DNA replication?
-initiation of DNA replication occurs by the assembly of a pre-replicative complex (pre-RC) at each potential origin of DNA replication by the sequential binding to DNA of the origin recognition complex (ORC) and cell division cycle 6 (Cdc6) that then recruit chromatin licensing and DNA replication factor 1 (Cdt1) that chaperones the DNA helicase mini-chromosome maintenance proteins2-7 (MCM2-7), forming ORC/Cdc6/Cdt1/MCM2-7 (OCCM) complex
-complete assembly of the pre-RC involves the recruitment of a second Mcm2-7 hexamer to form head-to-head Mcm2-7 hexamers that are destined to become the core components of the DNA helicases at the two replication forks that emanate from each origin
what are the following steps of DNA replication?
-activation of pre-RCs during S phase results in their destruction. Hence, re-initiation cannot occur on replicated DNA
-origin licensing is restricted to the G1 phase of the cell cycle when S phase cyclin-dependent kinase (CDK) activity is absent. Active CDK phosphorylates pre-RC components and inhibits pre-RC assembly, and hence, the pre-RC assembly cannot occur once cells enter into S phase and can only re-occur after cells pass through mitosis when CDKs are destroyed
-phosphorylation of ORC, Cdc6, and Mcm2-7 protein by CDK control pre-RC assembly. The same CDK activity that prevents pre-RC assembly is activated just before S phase and is required for the initiation of DNA synthesis at each origin
how does replication occur?
-pre-IC (initiation complex) formation is a subsequent stage following the pre-RC assembly, in which licensed origins initiate replication in a temporarily regulated manner throughout S phase
-the pre-IC is formed by recruiting additional factors including Cdc45, GINS, Sld7, Sld7, Sld3, Sld2, Dpd11, Pol e, RPA and Mcm 10 to the Mcm2-7 double hexamer, which activates 3’ to 5’ helicase activity. Single-stranded DNA-binding (SSB) protein (replication protein A/RPA), helix-destabilizing proteins, ssDNA protection from re-annealing and nucleases
what are DNA polymerases (replication 2)?
-DNA polymerase alpha-primase is recruited to prime the initiation of replication and DNA polymerases drive the the bi-directional replication forks, where Pol e is dedicated to the replication of the leading strand, whereas Pol & is dedicated to the replication of the lagging strand.
-DNA polymerase (5’ to 3’ polymerization but 3’ to 5’ exonuclease activity) on the leading strand
-lagging-strand synthesis leads to the formation of single-stranded DNA (ssDNA), which is coated with replication protein A (RPA)
-fork progression requires the activity of several replication cofactors, including the clamp proliferating cell nuclear antigen (PCNA)
-DNA unwinding by the replication fork generates positive supercoiling, which is alleviated by topoisomers
-replications also entails reassembly of recycled and de novo-synthesized nucleosomes at the newly synthesized DNA
what are the types of transcription replicative stress?
-head-on collisions
-co-directional collisions
-fragile sites
what are head-on collisions?
-progression in opposite directions of an RNA polymerase (RNAP) and a replication fork leads to head-on collisions, which induce the pausing and blockage of the replication fork, and may lead to its collapse and the formation of DNA breaks
what are co-directional collisions?
-progression of an RNAP and a replication fork in the same direction leads to co-directional collisions if the fork moves more quickly than the RNAP
-co-directional collisions can be resolved by displacement of the RNAP from the DNA
what are fragile sites?
-fragile sites are genomic regions that exhibit constrictions or gaps in metaphase chromosomes following replication stress
-they are categorized into into classes: rare fragile sites, which are found in <5% of individuals and arise from trinucleotide repeat expansion and common fragile sites (CFSs), which are found in all individuals and are not associated with repeat expansion
-early replication fragile sites (ERFSs) bind to ssDNA
what are the types of abnormal DNA structures?
-non-B DNA
-supercoiling
-hairpins
-triplex DNA
-G-quadruplexes
-gammaH2AX foci
what is non-B DNA?
not normal DNA
-any DNA structure that is different from right-handed double helix with 10 nucleotides per turn
what is supercoiling DNA?
over or under winding of the DNA helix
what is hairpin DNA?
DNA structures in which a strand folds on itself and forms intra-strand base pairing
what is triplex DNA?
a single-stranded DNA region bound to the major groove of the DNA duplex forming a three-stranded helix, normally at sequences with mirror symmetry
what are G-quadruplexes?
four repeats of at least three guanines that can interact to form four-stranded DNA structures
what are the DNA repair pathways?
-translesion synthesis (TLS)
-PrimPol-mediated repriming
-template switching (TS)
-break-induced replication (BIR)/nonhomologous end joining (NHEJ)
-homologous recombination (HR)
what is translesion synthesis?
direct replication of DNA damage through polymerases
what is PrimPol-mediated repriming?
DNA damage tolerance enzyme possessing both primase (ie: replication restart) and polymerase (replication) activites
what is template switching?
use the newly synthesized strand as the template to avoid the lesion
what are direct DNA breaks in single-strand?
-direct DNA breaks (which may arise from radiation, oxidation or abortive Top1-DNA intermediates) detected by poly ADP ribose polymerase (PARP)
what is DNA base excision repair (BER)?
what is nucleotide excision repair (NER) (single strand)?
what is double strand repair?
-homologous recombination repair (HRR)= error-free, critical for radio-sensitivity
-non-homologous end joining (NHEJ/BIR)= error-prone
what are the difference on NHEJ and HRR?
ATM= recognize double strand break. bound by p53 (governs cell death) which blocks nonhomologous recognition
what is cross-like repair? (double)
DNA-DNA or DNA-protein as in actively transcribed genes
what is transcription-replication collision prevention?
what is mismatch repair?
recognize replisome
what is more DNA repair machinery?
what is DDR?
what is clustered DNA damage by low energy electrons?
what can DNA cluster lesions be quantified by?
how common are double strand breaks?
what are the two types of aberrations?
-lethal (unstable, cause reproductive death)
-non-lethal (stable >50 years, but carcinogenic)
what is the graph of an example of anaphase bridge aberration?
what is the graph of rings, dicentric and anaphase bridge aberrations?
what is the graph of symmetric translocation and insertional deletion aberrations?
what is the graph of lymphocyte radio-sensitivity?
