Exam 2: Lecture 4

Question 1

Replication Origins

Answer 1

• access to single stranded DNA templates must be granted to the replication machinery to replicate chromosomes
• involves opening of the double helix at positions that are called replication origins
• as replication bubble forms, DNA synthesis will proceed bi-directionally through the opposite movement of two replication forks.
• each newly synthesized strand of DNA comprised of leading and lagging strands due to orientation of polymerase proteins in relation to movement of replication forks

Question 2

Replication in E. coli

Answer 2

• initiates at a single sequence-specific element
• two replication forks move rapidly away from each other and through the bacterial chromosome thus allowing for a ~30min cell cycle

Question 3

Replication in Human Genome

Answer 3

• 700-fold greater in size but the replication forks move at a rate that is about 20-fold slower
• if there was only a single replication origin per chromosome then it would take 20 days for a single division to occur
• solution: gets cell division down to an hour, there are between 30,000 and 50,000 replication origins used at each cell cycle

Question 4

Location of Origins

Answer 4

• not possible to accurately predict these in higher organisms
• also, has been shown that potential replication origins lie in excess and that only a subset are used during any one cell cycle
• general case that more replication origins are used during embryonic cell divisions than during subsequent cell cycles
• also more origins used under stress conditions

Question 5

Features Associated With Replication Origins

Answer 5

• even though there up to 50,000 potential origins in some genomes (humans) not all of them are used at each cell cycle (similar in yeast)
• selective use of particular origins must be dependent upon factors beyond a consensus sequence
• at sequence (nucleotide) level presence of AT rich regions of CpG islands are associated with replication origins
• chromatin structure can also influence the use of replication origins
• areas devoid of nucleosomes or are looped are commonly associated with replication origins
• promoters and transcriptional start sites also serve frequently as replication origin sites because these areas are in a state that is accessible to DNA binding proteins

Question 6

Classes of Replication Origins

Answer 6

• one consists of “consecutive” origins: these are bound by preRC machinery and are functional in all cell types and at all developmental stages
• second consists of “inactive or dormant” origins: never used despite being bound by preRC machinery
• last are “flexible” origins: always bound by preRC machinery but are functional only in certain cell types and developmental stages
• molecullarly it’s hard to tell the difference between flexible and inactive origins

Question 7

Origin Selection and Cell Cycle

Answer 7

• during normal cell cycle previously used replication origins are deactivated during mitosis
• halfway through G1 cells reach point called Origin Decision Point or ODP
• location of subset of origins that will be used in next cell cycle are determined at this point

Question 8

Origin Selection During Early Embryonic Development

Answer 8

• in both vertebrate and drosophilia a modified cell cycle called rapid embryonic oscillator is used
• this version quicky oscillates back and forth without G1 phase (G2 also missing in many organisms)
• normal somatic cell cycle previous used origins are inactivated during M phase
• since this type lacks G1 phase the cell does not specify which origins will be used in the next cycle and remain unspecified thus the S phase cell has wider range of potential orgins to choose from
• this is why embryonic cells use a greater number of origins than somatic cells

Question 9

Origin Selection During Endocycle

Answer 9

• cells oscillate between S and G1 and do not undergo mitosis
• origins deactivated in M phase so cells that are going through an endocycle do not deactivate their replication origins
• same origins are used at each cell cycle until cell stops using the endocycle and starts using the normal somatic cycle

Question 10

Licensing of Replication Origins

Answer 10

• happens in G1 phase
• however all origins not activated (replication bubble formation) at the same time
• some sections of genome replicated early in S phase while others are replicated during mid and late S phase
• reasons why are unknown
• often times origins activated early in S phase are found in clusters as are those activated late in S phase which is likely influenced by local state of chromatin structure (nucleosome rich or poor)

Question 11

Cell Cycle Regulators

Answer 11

• used to suppress the formation of new pre-replication complexes during S, G2 and M phases once replication has initiated from licensed origin
• these regulators include a subset of cyclin-dependent kinases (Cdk)
• this prevents inappropriate re-replication of chromosomal segments
• exception to rule takes place during endocycle-during this variation of the cell cycle the cell is attempting to replicate its genome (or parts of its genome) more than once
• when one origin fires, usually neighboring origins fire as well
• sites are used in some sequential order but it is unknown why

Question 12

Replication Initiation

Answer 12

• multistep process
• first step involves binding of heterohexamer protein complex called Origin Recognition Complex (ORC) which is the only known protein that actually recognizes and binds to the replication origin directly
• after ORC is bound to origin set of additional proteins including CDC6, CDT1, MCM9, and MCM2-7 (DNA helicase hexamer) hexamer are recruited
• binding of all these factors to replication origin marks the formation of pre-replication complex (pre-RC) this step called “licensing” of orign
• next step formation of pre-initiation complex (pre-IC) which requires further recruitment of additional proteins such as CDC45
• once pre-initiation complex has formed then DNA primase and DNA polymerase can be recruited to the origin
• once DNA primase and DNA polymerase are brought to the origin they bind to DNA helicase (MCM2-7) and together this complex unwinds the double helix, lays down short primer sequences and begins synthesizing the leading and lagging strands

Question 13

pre-Replicative Complex (pre-RC) and Mutations in ORC

Answer 13

• consists of the six ORC proteins (ORC1-6) and several additional factors including CDT1, CDD6 and helicase hexamer MCM2-7
• mutations in ORC1, ORC4, ORC6, CTD1 and CDC6 are associated with differing types of diseases/syndromes

Question 14

Suppression of Re-Replication of Genome

Answer 14

• during S phase of the cell cycle, the genome must be replicated once and only once
• after replication has been initiated by ORC1-6, CDT1, CDC6 and MCM2-7, these proteins must be prevented from re-assembling at the replication origins
• after replication has initiated the only proteins that remain at the origin are ORC1-6
• MCM2-7 helicase proteins leave the replication origin and continue to unwind DNA at the replication fork
• Cdc6 is exported from the nucleus to the cytoplasm
• lastly, CDT1 is phosphorylated and then degraded
• these mechanisms prevent the pre-RC from re-assembling at the origin until the next S phase
• over-expression of CDT1 can cause the re-replication of DNA within a single S phase

Question 15

Re-Replication of Genome

Answer 15

• in addition to polyploidy, re-replication of the genome leads to several deleterious effects including genome instability, double stranded breaks and tumorigenesis
• failure of mechanisms that remove pre-RC from replication origins after initiation of DNA replication leads to re-replication
• during re-replication, there is an increase in the number of double stranded breaks (DSBs) throughout the genome
• cell attempts to repair these breaks by inducing the ataxia telangiectasia (ATM) and telangiectasia and Rad3 (ATR) checkpoint pathways which arrest the cell cycle and in some instances can induce cell death
• both outcomes can suppress harmful effects of re-replication
• if re-replication occurs in a cell that is defective in its ATM and ATR checkpoint pathways then this leads to genome instability (due to accumulation of double-stranded breaks throughout the genome and to tumorigenesis (and cancer))
• DSBs means broken chromosomes. So checkpoints have machinery to fix breaks. If too many breaks these checkpoints can trigger machines to bring on the death

Question 16

Plasmids

Answer 16

-naturally occurring small circular pieces of DNA that can be replicated independently of the bacterial chromosome

Question 17

Identification of Replication Origins (Plasmids)

Answer 17

• ability to be replicated is due to presence of a replication origin
• synthetic plasmids can be generated for many purposes including the identification of replication origins synthetic plasmids can be generated that lack replication origin
• such plasmids cannot be replicated within bacterial cells
• chromosomal DNA from a cell/tissue can be digested with a restriction enzyme to yield small fragments of DNA
• synthetic plasmid is made to contain a restriction enzyme recognition site which allows for the plasmid to be digested with the same enzyme that was used to digest the chromosomal DNA
• these enzymes will yield either blunt or sticky ends
• can mix the digested plasmid with the chromosomal fragments and glue/ligate them together with DNA ligase
• ligated circular plasmids can then be transformed into bacterial cells (remember that Frederick Griffith demonstrated that bacterial cells can be transformed)

Question 18

Synthetic Plasmids and Antibiotics

Answer 18

• engineered to harbor a gene that confers antibiotic resistance, which will be used as a selectable marker in experiment
• transformation efficiency of bacterial cells is fairly low so most of the bacterial cells will lack the circular plasmid
• need a way to get rid of these bacteria and only keep the ones that have the plasmid
• bacterial cell population will be added to a agar plate that contains an antibiotic
• if bacterial cell has not been transformed then it will die
• if it contains the plasmid it will survive provided that the plasmid has an origin of replication
• required to produce multiple copies of the antibiotic resistance gene which in turn is necessary for producing the protein that can counteract the effects of the antibiotic that has been added to the agar media
• if bacterial cell has a plasmid that lacks a replication origin it will not produce enough of the antibiotic resistance protein and will die
• if it has an origin, multiple copies of the plasmid (and thus the gene) will be made in the cell and the antibiotic resistance protein will be made in high enough levels to support bacterial growth on the agar plate
• will be manifested as a bacterial colony.
• the identity of the origin can be determined by sequencing