DNA Replication 8.21

Question 1

which residues are most commonly phosphorylated by kinases and why

Answer 1

serine, threonine, and tyrosine - have a readily accessible hydroxyl group

Question 2

What happens during G1 when the cell-cycle is induced

Answer 2

p27 (CDK inhibitor) - decreases and remains low cyclin-D production rises and is maintained throughout the remainder of the cell cycle

Question 3

where and what are the four checkpoints of the cell cycle

Answer 3

A. Cyclin-D/CDK-4 - determines whether the cell goes into G0 B. Cyclin-E/CDK-2 - the big restriction point between G1 and S phase C. Cyclin-A/CDK-2 - progression through S phase D. Cyclin-B/CDK-1 - between G2 and M phase

Question 4

Describe the first cell-cycle checkpoint

Answer 4

cyclin-D/CDK-4 growth factor enters the nucleus and binds myc (transcription factor) - initiates transcription of gene for cyclin D once translated, cyclin D is exported from the nucleus and binds CDK4 in the cytoplasm the activated hetero dimer phosphorylates retinoblastoma (Rb) which deactivates Rb deactivated Rb releases E2F (a transcription factor) E2F is free to activate transcription of genes for cyclin-E and cyclin-A

Question 5

Describe the second cell-cycle checkpoint

Answer 5

G1/S restriction point mediated by Cyclin-E/CDK2 after transcription of Cyclin-E, it binds to CDK2 in the cytoplasm p53 actively opposes this by transcribing the gene for p21, an inhibitor of cyclinE/CDK2 heterodimer formation if Cyclin-E activity is high enough, the heterodimer is formed and moves into the nucleus to initiate DNA replication in the S phase

Question 6

why is p53 relevant to cancer

Answer 6

p53 activates apoptosis, mutation in the p53 gene is a common cause of cancer

Question 7

Describe the 3rd cell-cycle checkpoint

Answer 7

carries the cell through the S phase cyclin-A/CDK2 dependent cyclin-A production was activated at the first checkpoint when E2F was released from Rb to activate transcription of cyclin-A and E genes cyclin-A is exported to the cytoplasm where it binds with CDK2 and moves into the nucleus the heterodimer phosphorylates replication complexes to activate them

Question 8

Describe the 4th cell-cycle checkpoint

Answer 8

allows cell to progress from G2 to M phase [cyclin-B production and binding to CDK-1 not explained] this checkpoint is dependent on heterodimer being dephosphorylated by cdc25 (phosphatase) activated heterodimer is moved into the nucleus and causes envelope breakdown, assembly of mitotic spindle, and metaphase arrest

Question 9

what are some characteristics of cancer

Answer 9

what are some characteristics of cancer

Question 10

what are the five gene groups involved in cancer

Answer 10

proto-oncogenes, tumor suppressor genes, genes regulating apoptosis, genes regulating senescence, and DNA repair genes

Question 11

What are proto-oncogenes? What are some common proto-oncogene related causes of cancer

Answer 11

Proto-oncogenes encode proteins involved in cell growth and proliferation - mutation causes production of proteins that are pathologically activated (never shut off), thus resulting in the formation of tumors EGFR (epidermal growth factor receptor) - non-small cell lung carcinoma Src - tyrosine kinase receptor - sarcoma; colon cancer Ras - GTPase - 25% of all human cancers Myc - (first step transcription factor) Cyclin-D - (first step cyclin)

Question 12

What are tumor suppressor genes and give some examples with their function

Answer 12

Genes that suppress the cell cycle - mutations can cause loss of function/expression p21: inhibits CDK-4 and CDK-2 p53: induces apoptosis and activates the gene encoding p21 Rb: binds E2F to inhibit cyclin-E and A BRCA-1 and BRCA-2 repair broken DNA

Question 13

Give examples of the function of genes which regulate apoptosis

Answer 13

Tumor Necrosis Factor (TNF) - released by macrophages - bind to TNF receptors on the cell surface TNF receptor concentration balances pro- and anti-apoptotic factors - when TNF receptors are sufficiently saturated, mitochondria will leak cytochrome C into the cytoplasm - this activates Caspase which fragments chromatin and thus disrupts the cell bcl-2 is a protein that inhibits apoptosis - mutations causes overactivation which results in tumor development - B Cell Lymphoma

Question 14

Explain the function of telomerase

Answer 14

maintains telomere length in embryonic stem cells and germline cells - typically inactive in somatic cells mutation results in constitutively active telomerase resulting in immortal cells

Question 15

how are DNA repair genes involved in cancer

Answer 15

genes like BRCA are responsible for DNA repair - mutation that causes loss of function allows mutations to accumulate

Question 16

How can cancers be preempted

Answer 16

test for BRCA mutations as an indicator of cancer risk test for cancer antigens to catch early stage cancer

Question 17

What are three main objectives in cancer treatment

Answer 17

Prevent metastasis - drugs can inhibit metallo-proteases that are needed for cells to metastisize target angiogenesis - prevent growth of blood supply to limit tumor size target specific molecules - inhibit growth factors, activate senescence or apoptotic genes, destroy pathogenic RNA’s

Question 18

what are the four basic properties of DNA replication

Answer 18

semiconservative - double stranded DNA contains one strand of parent and one strand of daughter DNA DNA is synthesized in the 5’-3’ direction A 3’-OH primer on the growing strand and a template parent strand are needed DNA replication is semi-discontinuous - on the lagging strand of DNA

Question 19

What are the three possible combination in which DNA can replicate

Answer 19

Conservative - parent strands end up back together, daughter strands end up together Dispersive - mixed parent and daughter sequences in each strand Semiconservative - parent on one strand, daughter on the complementary strand

Question 20

Why is a 3’-OH primer needed for replication

Answer 20

The -OH residue nucleophilically attacks the alpha phosphate of the dNTP, forming a new phosphodiester bond and kicking off a pyrophosphate (PPi)

Question 21

What is responsible for making the RNA primers that start Okazaki fragments

Answer 21

primase

Question 22

What are the four basic steps of DNA replication

Answer 22

Separation of the two strands at an origin of replication (prokaryotic DNA has 1 origin, human DNA has thousands) Formation of the replication fork (where primers for Okazaki fragments are placed as the DNA unwinds Chain elongation - new DNA being assembled Removal of RNA primers

Question 23

Describe the three functions of DNA polymerase I

Answer 23

1. polymerase - grow the DNA chain from 5’-3’ 2,3. exonucleases - cleave phosphodiester bonds from both the 5’->3’ and 3’->5’ directions as necessary - proofreading the DNA

Question 24

Differentiate between DNA polymerase I,2, and 3

Answer 24

DNA polymerase 1 and 2 synthesize small fragments (used to fill in fragments) - DNA polymerase 3 synthesizes much larger lengths of DNA (used for most of replication) All of them can proofread from 3’->5’ direction Only DNA polymerase 1 can proofread from the 5’->3’ direction - largely used in repair

Question 25

Name the proteins involved in replisomes and give their function

Answer 25

Single-stranded DNA binding protein (SSB) - keeps DNA from reannealing while replication is taking place Helicase - unwind DNA Primase - form RNA primers for the start of Okazaki fragments polymerase - assemble DNA ligase - seal breaks in the DNA (whether due to repair or to seal Okazaki fragments gyrase (a.k.a topoisomerase) - relax supercoiling

Question 26

Describe the chromosome end problem and telomerase’s role

Answer 26

primase has no issue placing a primer at the start of the lagging strand DNA polymerase however requires a 3’-OH residue to insert nucleotides and thus is unable to fill the first space left by the primer after it is stripped away telomeres are series of repeating TTAGGG sequences that don’t contribute to gene expression these grow shorter with each repetition and typically a cell will enter senescence once its telomeres are exhausted

Question 27

Describe the chromosome end problem and telomerase’s role

Answer 27

primase has no issue placing a primer at the start of the lagging strand DNA polymerase however requires a 3’-OH residue to insert nucleotides and thus is unable to fill the first space left by the primer after it is stripped away telomeres are series of repeating TTAGGG sequences that don’t contribute to gene expression these grow shorter with each repetition and typically a cell will enter senescence once its telomeres are exhausted

Question 28

Describe telomerase’s mechanism

Answer 28

Telomerase is an RNA dependent DNA polymerase - it uses an internal RNA sequence to partially bind to the template strand and then uses its polymerase capabilities to add onto the template strand it repeatedly extends the template strand with G-T residues and translocates further up the line after telomerase dissociates, primase lays down a primer and DNA polymerase fill in the Okazaki fragment - the remaining single stranded fragment is protected by TRF1 and TRF2 (telomere-binding proteins