Allingham Lecture 10- BCHM218

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BCHM 218 (Molecular Biology)

February 24, 2015

Lecture 10: DNA Replica9on

Dr. John Allingham Bo8erell Hall Rm. 652

Department of Biomedical and Molecular Sciences allinghj@queensu.ca

Please read MBPP, pp. 32-36, 364-372

Review of SecLon 1 and 2 We have covered the fundamental principles that determine the informaLon-storing and funcLonal capabiliLes of geneLc informaLon molecules:

The structures of protein and DNA building blocks, as well as genomes and gene products.

The mechanisms by which they are organized. Their chemical properLes and reacLvity. Methods with which to isolate, manipulate, and

study them.

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Preamble to SecLon 3 and 4 We will now use these principles to understand the molecular basis by which genomes are copied and edited, and geneLc informaLon is regulated:

Basis for DNA replicaLon delity. Mechanisms of geneLc recombinaLon. Processes involved in decoding DNA-based

messages into molecular devices that enable life.

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The EukaryoLc Cell Cycle

(two copies of each

chromosome)

(each chromosome is duplicated)

(four copies of each

chromosome)

(mitosis)

(quiescent)

Every Lme a cell divides, it replicates all of its DNA; a process that takes place in a ma8er of hours.

Eukaryote genomes are large - nearly 3 billion bases in the human genome!

Considering that the human body houses about 15 million, million cells, each one harboring a (near) perfect copy of the genome (two copies in fact), it is important that mistakes are minimized.

Some consideraLons:

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A large number of factors are associated with the process of DNA replicaLon to ensure rapid and faithful reproducLon of the geneLc material.

Some consideraLons:

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Beginnings of ReplicaLon Enzymology

Arthur Kornberg

How is the DNA polymer made?

Kornberg et al. followed the fate of radioacLve [14C]thymidine (ensured that whatever radioacLve polymer made was DNA, not RNA)

Puried DNA polymerase I (Pol I). Demonstrated that Pol I received

instrucLons on what nucleoLdes to incorporate from an exisLng template (parental DNA strands).

Marked the beginnings of biotechnology.

Pg. 403

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Enzymes and other components involved in replicaLon can be idenLed, puried, and have their funcLons analyzed biochemically.

Some Biochemical Principles

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A fairly simple way to observe the acLvity of a DNA polymerase is to measure the incorporaLon of a radioacLvely labeled deoxyribonucleoside or deoxyribonucleoLde into the high molecular weight polymer DNA. The la8er can be precipitated with a strong acid, such as trichloroaceLc acid, whereas unincorporated nucleoLdes or nucleosides do not precipitate.

For instance, a crude cell extract can be incubated with dTTP labeled with a 32P atom in the - phosphate (abbreviated [32P] dTTP) plus other unlabeled nucleoside triphosphates, appropriate buers and cofactors. The DNA synthesized in this reacLon can be measured as the amount of 32P precipitated by acid (Figure 5.9A).

Biochemical studies ogen involve an assay for the acLvity under invesLgaLon, which is usually a measurement of the product of the reacLon being catalyzed by the enzyme.

Some Biochemical Principles

Incorpora9on Assay: A simple way to observe the

acLvity of a DNA polymerase is to measure the incorporaLon of a radioacLvely or uorescently labeled dNTP into the high molecular weight polymer DNA.

Materials and Methods

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Detec9on of DNA polymer:

This can involve precipitaLon of the polymer with a strong acid, such as trichloroaceLc acid (unincorporated nucleoLdes or nucleosides do not precipitate).

Analyze radioacLvity by Geiger counter, scinLllaLon counLng, or electrophoresis and autoradiography.

Materials and Methods

[32P] dTTP

incorporation

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Detec9on of DNA polymer:

This can alternaLvely involve use of a posiLvely charged lter (in low salt) to which negaLvely charged polynucleoLde backbone will bind, but unincorporated nucleoLdes will pass through.

Analyze uorescence signal by uorescence detector.

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IdenLcaLon and isolaLon of the DNA polymerizing acLvity from other proteins can be done by chromatography.

Each fracLon from a chromatographic column is assayed for protein (e.g., the absorbance at 280 nm, gray line) with the ability to catalyze the incorporaLon of [32P] dTTP into DNA (black line).

Materials and Methods

RadioacLvity

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PuricaLon of Polymerase by Kornberg et al. involved a mulLstep process in which protein concentraLon and specic enzyme acLvity (DNA polymerizaLon) were monitored.

Results

(Lehman, I. R., Bessman, M. J., Simms, E. S., and Kornberg, A. (1958) J. Biol. Chem. 233, 163170)

Requirements of the PolymerizaLon ReacLon were deduced by Kornberg et al. by showing that the maximal incorporaLon of labeled deoxyribonucleoLdes into DNA (monitored by amount of precipitated DNA with dTP* incorporated) was dependent on the presence of polymerized DNA, Mg++, and the deoxynucleoside triphosphates of thymine, cytosine, guanine, and adenine.

Results

(Bessman, M. J., Lehman, I. R., Simms, E. S., and Kornberg, A. (1958) J. Biol. Chem. 233, 171177)

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Conclusions: DNA Polymerase ReacLon Components

Requirements: All DNA polymerases require a template strand. All DNA polymerases require a primer strand.

Deoxyribonucleoside 5-triphosphates (dNTPs)

Polymerase enzyme + associated cofactors

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These two classics were declined by the JBC when submitted in the fall of 1957. Among the critical comments were:

It is very doubtful that the authors are entitled to speak of the

enzymatic synthesis of DNA; Polymerase is a poor name

More consideraLons:

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Is DNA replicaLon a new invenLon?

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DNA replica9on apparently evolved twice.

Koonin and MarLn: From detailed gene sequence comparisons

Eugene Koonin found that bacteria and archaea broadly share the same mechanisms (and enzymes) of protein synthesis (i.e. how DNA is read into RNA, and how that RNA is then translated into proteins).

This is not the case for the enzymes needed for DNA replicaLon. Most have nothing at all in common between bacteria and archaea.

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Nucleic Acids Res. 1999 September 1; 27(17): 33893401

Allingham Lecture 7- MBIO218

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Viral

Bacterial

Viral

Human

DNA polymerases

Why Are There So Many Diverse Replication Machineries?

Journal of Molecular Biology, Volume 425, Issue 23, 2013, 4714 - 4726

Proteins and mechanisms of Okazaki fragment synthesis

Koonin and MarLn: There are, however, many important

funcLonal parallels among all known cellular systems (bacteria, archaea, and eukaryotes) of DNA replicaLon.

These common features can be roughly summarized as follows:

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(1) DNA replicaLon is semi-conservaLve

Each daughter chromosome contains one parental strand and one newly synthesized strand.

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(2) ReplicaLon is iniLated at specic sites

Origins of replicaLon are used in conjuncLon with an origin recogniLon system. 25

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(3) ReplicaLon is typically bidirecLonal

ReplicaLon fork moves away from the origin of replicaLon in both direcLons.

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(4) ReplicaLon is conLnuous on the leading strand and disconLnuous on the lagging strand

Both daughter strands cannot be replicated in the same direcLon that the replicaLon fork moves.

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(5) RNA primers are needed to start DNA replicaLon

Primer strands must be complementary to the template and contain a free 3-OH group.

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(6) nucleases, polymerases and ligases replace the RNA primers with DNA and seal the remaining nick

KEY CONVENTION The direcLon of synthesis by DNA polymerases refers to the direcLon in which each new nucleoLde is chemically linked to the growing daughter strand. All DNA polymerases funcLon in the 53 direcLon, linking the -5-phosphate of a new dNTP to the 3 posiLon of the nucleoLde residue at the end (i.e., the 3 end) of the chain.

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