Molecular Biology Ch14-16 (Fall 2021- Petrik)
Ruv - of the RuvAB complex is responsible for --; Ruv - of the RuvAB complex is responsible for --. RuvC performs --.
A; holding the Holliday junction open; B; branch migration; cleavage to resolve the Holliday junction
-- activation due to sensing a DNA double stranded break triggers this regulatory kinase to phosphorylate the histone variant protein -- to form modified chromatin on either side of the DSB. This causes phosphorylation by ATM of --, which amplifies the activation of ATM. The phosphorylation of the 2 above proteins provides a platform for large complexes of regulatory and repair proteins. Phosphorylation of -- promotes HR (homologous recombination), whereas phosphorylation of -- promotes NHEJ (Non-Homologous End Joining).
ATM; H2AX; MDC1; BRCA1 and CtIP; 53BP1
Eukaryotic proteosomes degrade proteins that are no longer needed by the cell. They are shaped like a barrel, with the___________ domain on the outside that feeds the protein into the barrel. The _________ domain (also known as a protease domain) is on the inside of the barrel, and degrades the protein into 5-20 amino acid sized pieces/ (fill in the blank)
ATPase Peptidase
In this pathway showing gene conversion between yeast mating types, how does the silent alpha mating type end up getting expressed?
After a double stranded break and resection at the MAT site, the silent alpha sequence at the HML site is used as a template for homology directed repair at the MAT site
Why is the usual/most frequently used DNA double stranded break repair pathway available in G1 of the cell cycle the NHEJ pathway, not the HDR pathway?
Because the cell-cycle regulatory kinase CDK1, that activates CtIP needed for generation of 3' single stranded ends, is only active in S and G2
If a DNA lesion occurs at a replication fork, regression of the fork leads to pairing of the newly synthesized strands, and pairing of the parental strands. DNA repair then occurs either by base and nucleotide excision repair occurs. Any one of the these three mechanisms can then allow a normal replication fork to be restored: --, --, --.
Cleavage and fork restoration; degradation of paired newly synthesized strands; reversal of regression
How does Eukaryotic double stranded break sensing (before repair) trigger arrest in the cell cycle until the repair is complete?
DNA damage sensors phosphorylate check point kinases, which indirectly inactivate cyclin dependent kinases that promote transition through the cell cycle.
Match which details pertain to homology directed repair (HDR), and which pertain to homologous recombination (HR) If two different alleles are present across homologous chromosomes and a DSB is repaired by HDR using the homologous chromosome as template- what can occur? Homologous recombination can result in The newly synthesized strand is paired back to the 3' end of the other broken DNA strand The displaced template strand of the D loop pairs with the other broken strand and serves as a template for DNA synthesis- forming a Holliday junction
Gene conversion at the DSB site but no rearrangement of gene alleles surrounding the DSB site Genetic recombination of alleles of genes flanking the DSB site Homology directed repair Homologous recombination
What is the common cause of gene conversion (which occurs rarely) during homology directed repair?
Gene conversion from one allele to another occurs when a homologous chromosome containing a different allele of the gene is used as the template to repair a double stranded break on its homolog. This occurs rarely compared to NHEJ during the G0, G1 and early S phases.
Which of the following is NOT a function of chaperone proteins?
Guide appropriate folded proteins to their correct cellular location
What is the cause of slipped strand misreplication, what does it cause; and how is it repaired?
Hairpin formation of repetitive DNA while it is in single stranded form in the replication bubble; causes insertions or deletions depending if it occurs on the template or new strand; repaired by mismatch repair systems
Fill in the blanks of the mammalian NHEJ pathway:1) A double stranded DNA break and degradation occurs2) The ends of the DSB are partially resected by nuclease, making short single stranded overhangs3) ________ heterodimers bind the broken ends and recruit __________ 5) The blank 3 answer trims the single stranded tails6) DNA ends are ligated by ________ (use roman numerals in part of the answer) in complex with XRCC4 and XLF/Cernunnos7) Ligation results in deletion
KU DNA-PKcs Artemis Ligase IV
Put the following steps of E. coli MutSLH mediated post-replicative mismatch repair in chronological order
MutS binds to the DNA mismatch MutL and MutH assemble with MutS at site of mismatch in the newly synthesized DNA The second lobe of MutL interacts with MutH at a hemi-methylated GATC site- leading to the formation of the MutSLH complex MutH is activated and nicks the newly synthesized unmethylated strand at the location of the methylated GATC on the methylated strand DNA including the mismatch is degraded by an exonuclease assisted by a helicase Re-synthesis of the unmethylated strand occurs correcting the mismatch
Answer these basic questions about nonhomologous end joining (NHEJ) and homology directed repair (HDR) of double stranded DNA breaks. Only DSB repair pathway available in G1 of the cell cycle when no homologous DNA spanning the DSB is nearby DSB repair pathway that uses either DNA from a sister chromatid or rarely DNA from a homologous chromosome as a template for repair Nucleases or polymerases remove a few bases on either side of the DSB and then religate Resection from the site of the break to reveal 2 single stranded regions with microhomology that align and then are ligated
NHEJ HDR Less frequent mechanism of NHEJ More frequent mechanism of NHEJ
If a replication fork encounters a DNA lesion that hasn't been repaired yet, translesion DNA synthesis can allow DNA replication to proceed. Why can translesion DNA polymerases traverse damaged DNA, and what is the consequence of this?
They have a more open active site; they are more error prone
Ubiquitination and sumoylation are the addition of small -- to the -- amino acids of proteins. Ubiquitination has roles in targeting proteins for -- and --, as well as trafficking and transcriptional regulation.
proteins; lysine; proteosomal degradation; mediating cellular response to DNA damage.
What are inteins? Often inteins are able to move around the DNA sequence encoding them into other parts of the genome. How do they do this?
A domain of a single polypeptide folded protein that is able to catalyze its own excision; its endonuclease domain is able to excise the DNA sequence encoding it and insert it into other genes that lack the intein sequence
Which of these is NOT a reason for chromosomal rearrangements as a result of homologous recombination
A missing homologous chromosome
Initial binding of ribosome associated proteins occurs co-translationally, and is done to --. In bacteria, the ribosome associated chaperone is known as --. Later protein folding is performed by -- because they were initially found to be strongly induced under high temperatures. -- is monomeric and acts like a clamp holding a hydrophobic segment of the protein, while -- chaperones form oligomeric barrel like structures with detachable "lids".
Prevent incorrect initial folding; trigger; heat shock proteins; HSP70; HSP60
In bacteria, when a double stranded break is present, the -- complex uses helicase and nuclease activity to expose 3'-ended single stranded DNA. Then the -- protein coats one exposed strand to encourage its invasion into a complementary duplex to form a D loop.
RecBCD; RecA
Match the definitions to the terms pertaining to base excision repair Base excision repair DNA glycosylase AP endonuclease DNA polymerase DNA ligase
Removal and replacement of single nucleotides recognizes and removes a damaged DNA base of a nucleotide Recognizes the nucleotide lacking a base and cleaves there Replaces the excised nucleotide Seals the nick in the sugar-phosphate backbone
Match the role of each enzyme involved in nucleotide excision repair of a bulky DNA lesion UvrAB complex UvrB UvrC UvrD DNA polymerase and ligase
Reognizes bulky DNA lesion opens the DNA around the lesion site Its endonuclease activity cuts the DNA backbone on either side of the lesion Removes damaged DNA Re-synthesis of excised DNA and ligation to seal the nicks
Put the following steps of Homology Directed Repair (HDR) in chronological order
Specialized helicases and nucleases resect the 5' ends of the broken molecule at least 50 nucleotides The single stranded DNA from one end of the damaged duplex locates and pairs with its complementary strand in the related unbroken duplex to form a D loop DNA synthesis off of the broken strand in the D loop occurs as the replication bubble translocates The newly synthesized strand is recaptured by the other broken strand of the DSB The second broken strand uses the newly synthesized strand as template for conservative DNA replication Ligation seals the nicks in the sugar phosphate backbone on each strand
Which do you think is the most frequent way that a gene product (a not yet post-translationally modified protein) can obtain different biological functions in different cell types via cleavage?
The genes for the cleavage enzymes are differently expressed across different cell types
What is the error that occurs in break-induced replication during homology directed repair?
The newly synthesized strand fails to pair with the other side of the double stranded break, and instead synthesis continues on the same template strand.
Put the steps of the bacterial SOS response pathway in chronological order
The replication fork stalls due to DNA damage at the replication fork RecA protein binds to singe-stranded DNA and becomes activated RecA cleaves LexA repressor that is bound to operator of SOS genes SOS repair proteins including RecA and DinI are produced SOS proteins repair DNA and RecA is inhibited by DinI so RecA comes off DNA LexA binds to operator of SOS genes thereby turning off their transcription
Define the below types of mutations in DNA with their causes or consequences Tautomer Transition mutation Transversion mutation hydrolytic depurination pyrimidine dimers deamination alkylation intercalation frameshifting
The transient non-enzymatic alteration of a nucleotide by changing of position of one of the hydrogen atoms that mediate base pairing If a pyrimidine base is replaced by another pyrimidine base or a purine base is replaced by another purine base If a pyrimidine base is replaced by a purine base or vice versa The hydrolytic breakage of the glycosidic bond between a purine and the sugar-phosphate backbone resulting in loss of purine base part of nucleotide Promoted by UV exposure of the DNA. DNA polymerase cannot proceed through a dimer so insertions or deletions are generated in the newly synthesized DNA A change in the base structure by removal of an amino group from cytoside which converts it to uracil Altering the structure of the base part of the nucleotide by adding ethyl or methyl groups which alters base pairing intercalation inserts into the DNA helix thus pushing the base pairs apart. Upon DNA replication the base pair interactions are altered or deletions are generated Caused by DNA insertions or deletions in protein coding regions
What is the most common reason that proteins are modified with lipids? How and where are these lipid groups normally added on the protein?
To target proteins to a cell membrane to enter into the hydrophobic lipid bilayer; covalently bonded at the N- or C- termini
Match the following names of lipid modifications to proteins with their definitions acylation prenylation GPI anchors
addition of fatty acyl groups onto the N- or C- termini of proteins addition of isoprenoid groups onto C-terminal cysteine groups composed of carbohydrate and fatty acid moieties and attached to the C-terminus of the protein
Glycans are attached to proteins by either N-linked or O-linked glycosidic bonds. What does N-linked or O-linked mean? Select the two correct answers. abN-linked means that the glycan is covalently bonded to the amido nitrogen group of asparagine amino acid side chains on the protein. eO-linked means that the carbohydrate is covalently bonded to oxygen of serine or threonine side chains on the protein N-linked means that the glycan is hydrogen bonded to the amido nitrogen group of asparagine amino acid side chains on the protein b N-linked means that the glycan is covalently bonded to the amido nitrogen group of asparagine amino acid side chains on the protein. c N-linked means that the glycan is covalently bonded to the N-terminus of the protein d O-linked means that the carbohydrate is hydrogen bonded to the oxygen of serine or threonine side chains on the protein e O-linked means that the carbohydrate is covalently bonded to oxygen of serine or threonine side chains on the protein f O-linked means that the carbohydrate is covalently bonded to the oxygen present at the C-terminus of the protein
b. N-linked means that the glycan is covalently bonded to the amido nitrogen group of asparagine amino acid side chains on the protein. e. O-linked means that the carbohydrate is covalently bonded to oxygen of serine or threonine side chains on the protein
During meiosis, and sometimes mitosis, Holliday junctions lead to genetic recombination.The length of Holliday junction -- affects how much DNA is recombined with the other chromosome. During meiosis (and rarely mitosis), there is/are -- Holliday junction(s) formed. If the/these Holliday junction(s) are resolved in the -- direction, no recombination occurs; but if it/they are resolved in the -- direction then recombinant molecules result.
branch migration; two; same; opposite
Protein glycosylation is the addition of glycans to proteins. What are glycans? Glycans are usually hydrophilic, so what effect do they usually have on protein solubility?
carbohydrate complex sugars; increase solubility
Disulfide bonds can covalently link pairs of -- residues, and allow these residues to exist stably in an oxidized form. Because the cytoplasm is a reducing environment, disulfide bonds are most often not found in -- proteins, but rather in -- proteins.
cysteine; cytosolic; secreted
Although homology directed repair often leads to gene conversion, rarely resolution of a Holliday junction during homologous recombination can instead result in gene conversion. What additional process needs to occur prior to resolution of the Holliday junction in order for gene conversion to take place?
mismatch repair pathway
Non-enzymatic modification of proteins, such as --, can be regulatory or detrimental.
nitrosylation and oxidation
In order for a Holliday junction to be dissolved (dissolution rather than resolution), resulting in --, three things are needed: a helicase, a --, and accessory proteins.
no genetic recombination; a topoisomerase
Ribosomal proteins must be associated with rRNA. In order for this to happen, the ribosomal proteins produced in the cytoplasm must be transported into the nucleus to associate with rRNA and then transported back into the cytoplasm. These proteins are recognized based on recognition of their -- by nuclear --, which will allow their transport through the nuclear pore complex. Inside the nucleus, -- will promote release of the import receptor from the protein. In the case of export of proteins from the nucleus, the protein's -- is recognized by a --, which carries it and associated Ran-GTP through the nuclear pore complex. Once in the cytoplasm, -- allows release of the protein.
nuclear localization signal (NLS); import receptors; Ran-GTP; nuclear export signal (NES); export receptor; GTP hydrolysis
Match the details to the questions asked on the left Chemical means of transferring information in the euk. DNA damage cascade The principal regulator kinase that responds to double stranded DNA breaks in euk's The principal regulator kinase that responds to stalled replication forks and other damage that leads to accumulation of single stranded DNA Effect of ATR activity on the rate of DNA replication Effect of ATR activity on the activity of trans-lesion DNA polymerases
phosphorylation ATM ATR slows DNA replication Increases activity of trans-lesion DNA polymerases
Protein phosphorylation, acetylation, and methylation are -- reactions, occur -- in response to stimuli, and alter a protein's activity, or --.
reversible; quickly; localization
Proteins that must be moved across a membrane have a -- at either their N terminus or their C terminus. This allows them to be recognized by -- (which are composed of both RNA and protein), and a -- protein which has a receptor domain and a motor. Once the protein has reached the outside of the membrane, it is transferred to a -- protein with channel that undergoes a conformational change to let the protein inside the organelle, for example.
sorting motif; signal recognition particle; transporter; translocon
Programmed cell death (which occurs when DNA is not repaired) is mediated by p53, a -- regulator. The level of p53 is highly controlled, most often by --.
transcriptional; post-translational modification such as protein ubiquitination or phosphorylation or acetylation
In eukaryotes, proteins are targeted to the proteasome by being --. In contrast, bacterial proteins can be targeted to the proteasome by unfolding of their --, which may be bound by a/an -- to mediate the interaction of the protein with the proteasome.
ubiquitinated; recognition sequences; adaptor protein
