Mary K. Campbell Shawn O. Farrell http://academic.cengage.com/chemistry/campbell

Chapter Ten Biosynthesis of Nucleic Acids: Replication

Paul D. Adams • University of Arkansas

Replication of DNA

• Naturally occurring DNA exists in single-stranded and double-stranded forms, both of which can exist in ________ and ________ forms • Difficult to generalize about all cases of DNA replication • We will study the replication of ________ ________ ________ DNA and then of ________ ________ ________ DNA • most of the details we discuss were first investigated in ________, particularly ________ ________

Flow of Genetic Information in the Cell

• Mechanisms by which information is transferred in the cell is based on the “________ ________”

Prokaryotic Replication

• Challenges in duplication of ________________ double-stranded DNA • achievement of continuous ________ and separation of the two DNA strands • ________ of ________ portions from attack by ________ that attack single-stranded DNA • synthesis of the DNA ________ from one 5’ -> 3’ strand and one 3’ -> 5’ strand • efficient protection from ________ in replication

Prokaryotic Replication (Cont’d)

• • Replication involves ________ of the two original strands and ________ of two new daughter strands using the ________ strands as ________

_________________ replication:

each daughter strand contains one template strand and one newly synthesized strand • Incorporation of isotopic label as sole _________ source ( 15 NH 4 Cl) • Observed that 15 N-DNA has a higher density than 14 N-DNA, and the two can be separated by density-gradient ______________

Which Direction does Replication go?

• DNA double helix unwinds at a specific point called an ______________________________ • Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is _______________ in most organisms • At each origin of replication, there are two _________ _____________, points at which new polynucleotide chains are formed • There is ______ origin of replication and ________ replication forks in the circular DNA of prokaryotes • In replication of a _______________ chromosome, there are several origins of replication and two replication forks at each origin

DNA Polymerase

• DNA is synthesized from its 5’ -> 3’ end (from the 3’ -> 5’ direction of the template) • the ________

strand

is synthesized continuously in the 5’ -> 3’ direction toward the replication fork • the ________

strand

(________ fragments) is synthesized __________ also in the 5’ -> 3’ direction, but away from the replication fork • lagging strand fragments are joined by the enzyme

DNA

___________

DNA Polymerase

DNA Polymerase Reaction

• The 3’-OH group at the end of the growing DNA chain acts as a _______________.

• The ______________ adjacent to the sugar is attacked, and then added to the growing chain.

Properties of DNA Polymerases

• There are at least _______ types of

DNA polymerase

(Pol) in

E coli

, _______ of which have been studied extensively

Function of DNA Polymerase

• DNA polymerase function has these requirements: • all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP • Mg 2+ • an _________ - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication • DNA-Pol I: repair and patching of DNA • DNA-Pol III: responsible for the polymerization of the newly formed DNA strand • DNA-Pol II, IV, and V: proofreading & repair enzymes

Supercoiling and Replication

•

DNA

_________ (class II topoisomerase) catalyzes rxn involving relaxed circular DNA: • creates a _______ in relaxed circular DNA • a slight unwinding at the point of the nick introduces _______ • the nick is _______ • The energy required for this process is supplied by ____________________

Replication with Supercoiled DNA

• Replication of supercoiled circular DNA •

DNA gyrase

has different role here. It introduces a nick in supercoiled DNA • • a _______ point is created at the site of the _______ • the gyrase opens and _______ the swivel point in advance of the replication fork • the newly synthesized DNA automatically assumes the ___________ form because it does not have the nick at the swivel point • _______ , a helix-destabilizing protein, promotes unwinding by binding at the replication fork

single-stranded binding (SSB) protein

_______ single-stranded regions by _______ tightly to them

Primase Reaction

• The primase reaction • RNA serves as a _______ in DNA replication • • _______ activity first observed

in-vivo

.

Primase -

catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence • __________ and _________ of new DNA strands • begun by DNA polymerase III • the newly formed DNA is linked to the 3’-OH of the RNA primer • as the replication fork moves away, the RNA primer is removed by DNA polymerase I

Replication Fork General Features

DNA Replication in Prokaryotes

• DNA synthesis is bidirectional • DNA synthesis is in the 5’ -> 3’ direction • the leading strand is formed continuously • the lagging strand is formed as a series of Okazaki fragments which are later joined • Five DNA polymerases have been found to exist in

E. coli

• Pol I is involved in synthesis and repair • Pol II, IV, and V are for repair under unique conditions • Pol III is primarily responsible for new synthesis

DNA Replication in Prokaryotes

• Unwinding • DNA gyrase introduces a swivel point in advance of the replication fork • a helicase binds at the replication fork and promotes unwinding • single-stranded binding (SSB) protein protects exposed regions of single-stranded DNA • Primase catalyzes the synthesis of RNA primer • Synthesis • catalyzed by Pol III • primer removed by Pol I • DNA ligase seals remaining nicks

Proofreading and Repair

• DNA replication takes place only once each generation in each cell • Errors in replication (

mutations

) occur spontaneously only once in every 10 9 to 10 10 base pairs • • Can be lethal to organisms

Proofreading

- the removal of incorrect nucleotides immediately after they are added to the growing DNA during replication (

Figure 10.10

) • Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 10 4 to 10 5 base pairs

Proofreading Improves Replication Fidelity

• ____________________: catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides • ______________________

:

Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity • ______________________

:

enzymes recognize that two bases are incorrectly paired, the area of mismatch is removed, and the area replicated again • ______________________

:

a damaged base is removed by DNA glycosylase leaving an AP site; the sugar and phosphate are removed along with several more bases, and then Pol I fills the gap

DNA Polymerase Repair

Mismatch Repair in Prokaryotes

• Mechanisms of mismatch repair encompass:

Eukaryotic Replication

• Not as understood as prokaryotic. Due in part to higher level of complexity.

• Cell growth and division divided into phases: M, G 1 , S, and G 2

Eukaryotic Replication

• Best understood model for control of eukaryotic replication is from yeast.

• DNA replication initiated by chromosomes that have reached the G 1 phase

Eukaryotic DNA Polymerase

• At least 15 different polymerases are present in eukaryotes (5 have been studied more extensively)

Structure of the PCNA Homotrimer

• PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (  ).

The Eukaryotic Replication Fork

The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Differences summarized in Table 10.5.

Telomerase and Cancer

(Biochemical Connections)

• Replication of linear DNA molecules poses particular problems at __________________ of the molecules • Ends of eukaryotic chromosomes called ______________________

:

__________________________________________ • See figures on p. 282-283