The Replication of DNA I

For the synthesis of DNA to proceed, we need __________ ________ and a ______ ________ _______

4 Examples of deoxynucleoside triphosphates

Nucleoside triphosphates have _____ _______ groups that are attached via the _________ of the 2'-deoxyribose. The group closest to the deoxyribose is called the _______, whereas the middle and distal are called the _______ & ______ respectively

The second essential substrate is a particular arrangement of ______ ______ ____ and ______ ______ ____ called a primer:template junction

In the primer:template junction, the template provides the ______ that directs the addition of each _______ _______. The primer is _______ to but _______ than the template.

The primer must have an exposed ______ adjacent to the single strand region of the ______. It is the _______ that will be extended by nucleotide addition

Why would we consider the primer the only substrate for DNA synthesis?

DNA is synthesized by extended which end of the primer? Is this feature uniform in both DNA & RNA

S_N2 reaction

The __________ bond is formed in an S_N2 reaction. The leaving group is _________ and it is composed of _______ & _______.

The template strand directs which of the _____ _______ _______ is added. The one that _____ ______ with the template strand is highly favored for addition to the ______ strand. This is facilitated by the ________ orientation of both strands.

The antiparallel orientation of both strands means that the ______ strand for DNA synthesis has the _______ orientation of the growing DNA strand

_______ of pyrophosphate into two _______ groups is the driving force for DNA synthesis. It is done by an enzyme known as ________. The remaining (Gibbs free) energy released during the reaction comes from?

DNA synthesis is catalyzed by a class of enzymes known as ____ ________ which use a single _____ _____ to complete the process

How are DNA polymerases different from most enzymes

How do DNA polymerases seemingly catalyze four separate reactions as opposed to just one?

The DNA polymerase monitors the ability of the incoming _______ to form an appropriate _____ _____ rather than detecting the exact _______ that enters the ______ site.

What happens when a correct base pair is formed by DNA polymerases? What happens when the wrong ones are formed? How much slower is the rate of formation of incorrect base pairs?

Kinetic proofreading

DNA polymerases show an impressive ability to distinguish between ribonucleoside and deoxyribonucleoside triphosphates (rNTPs and dNTPs). Although ______ are present at approximately _____ times the concentration in the cell, they are incorporated _____ times less than _____. This is mediated by the ______ exclusion of _____ from the DNA Polymerase active site.

In DNA polymerases the ______-______ pocket cannot accomodate the 2'OH on the incoming nucleotide. This space is occupied by two amino acids taht make ___ ____ _____ contact with the _____ ring. How would we make DNA polymerase less selective of rNTPs and dNTPS?

Nucleotides that meet some but not all of the requirements for use by DNA polymerase can inhibit DNA synthesis by terminating ________. Such nucleotides represent an important class of drugs to treat _____ & _____ infections

DNA polymerase looks like a _____ hand that grips the _____ _____ _____. The three domains of the polymerase are called the ______, _______, and ______.

The palm domain is composed of a ______ and contains the primary elements of the ______ ______. This region binds to two _______ metal metal ions, typically Mg2+ or Zn2+, that alter the chemical environment around the correctly _____ _____ ___ and the _____ of the primer.

How does one of the divalent metal ions affect the 3'OH end of the primer (2)

How does the 2nd of the divalent ions affect the pyrophosphate group (2)

The _____ domain monitors the base pairing of the most recently added nucleotides. This region of the polymerase makes extensive _______ _____ contact with base pairs in the ______ groove of the newly synthesized DNA.

The hydrogen bonds between the palm and base pairs is not _____ _____ specific. They only occur when nucleotides are _______ _____ ______

Three effects of mismatched DNA in the palm region & two possible results

Several residues in the fingers bind to incoming ______. Once a correct base pair is formed between the incoming _____ and the _______ the finger domain moves to enclose the ____. How does this stimulate catalysis?

The finger domain also associates withe the template region, leading to a nearly ___ degree turn of the ________ _______ between which two bases of the template

The 90° bend serves to expose only the _____ _____ base after the ______ at the catalytic site and avoids any confusion concerning which _____ _____ should be with the next nucleotide to be added.

Between the palm, fingers and thumb, which is the least involved in catalysis?

The thumb interacts with the DNA that has been most _______ synthesized. What two purposes does this serve?

The association between the thumb and _____ synthesized DNA contributes to the ability of the _____ _______ to add many ______ each time it binds a primer:template unction

The fingers of the polymerase closing around the base-paired dNTP places the critical catalytic _____ _____ in a position to catalyze formation of the next ________ _____. Attachment of the base paired nucleotide to the ______ leads to the reopening of the fingers and the movement of the _____ _____ ______ by one base pair. The _______ is then ready for the next cycle of addition.

The hand works best with accurate base pairing between the _______ _____ and the _______

Catalysis by DNA polymerase is ______, the polymerase is capable of adding as many as _______ nucleotides per second to a _____ strand. The speed of DNA synthesis is largely due to _______ nature of DNA polymerase.

Processivity
Degree of processivity (with regard to DNA polymerase)

Each DNA polymerase has a characteristic pprocessivity that can range from only ____ nucleotides to more than _______ bases per binding event

How to dramatically increase the rate of DNA synthesis?

It is the _____ binding of _______ to the primer:template junction that is rate-limiting for DNA synthesis.

How fast is the typical DNA polymerase reaction (3)

How fast is the fastest DNA polymerase reaction. What is the result compared to nonprocessive polymerases

Processivity is facilitated by ______ of DNA polymerases along the ____ ______. Once bound to a primer:template junction, ____ _______ interacts tightly with much of the double stranded portion of the DNA in a ______-_______ manner.

These sequence-nonspecific interactions include electrostatic interaction between the ______ _____ and the ______ domain and interactions between the ______ ______ of the DNA and the _____ domain. The sequence-independent nature of these interactions permits the _____ movement of the DNA even after it binds to ______.

Each time a nucleotide is added to the primer strand, the DNA partially releases from the ______, the ______ ______ with the minor groove are _______, but the _________ interactions with the ______ are maintained

After a nucleotide is added to the primer strand, DNA partially releases from the ______ and rapidly rebinds to it in a position that is shifted by ____ ____ _____ using the same _______-_______ mechanism. Increases in processivity are achieved through interactions between the DNA polymerase and ______ ______

Two limiting facts about the accuracy DNA synthesis

How does flickering of bases into wrong tautomeric form affect DNA synthesis?

Removal of incorrectly base paired nucleotides is mediated by a type of _______ that was originally identified in the same ________ as the DNA polymerase. It is called the ______ _______

Proofreading exonucleases are ______ that ______ DNA starting from the ____ end. Nucleases that only degrade from a DNA end are called ______; nucleases that can cut within a DNA strand are called ______.

The removal of mismatched DNA is facilitated by the ______ ability of DNA polymerase to add a nucleotide adjacent to an ______ base paired primer. Mispaired DNA alters the geometry between the _____ and the _____ _____ because of poor interactions with the ______ region.

The altered geometry resulting from mispaired bases reduces the rate of ______ ______ in much the same way that addition of an incorrectly paired dNTP reduces ______. Thus, when a mismatched nucleotide is added, it both decreases the rate of new _______ _______ and increases the rate of _________ ________ activity

As for processive DNA synthesis, proofreading occurs without _______ the DNA from the polymerase. When a mismatched base pair is present in the polymerase _____ _____, the primer:template junction is _________ creating several pairs of _______ DNA

The DNA polymerase active site binds such a mismatched template ______, but the exonuclease active site has a ____-fold higher affinity for ______ _______ _____. So, the newly unpaired 3'end moves from the polymerase active site to the ______ _____ ______ and the ______ ______ is removed by _______.

The removal of the mismatched base allows the primer:template junction to ______ and ______ to the polymerase active site, enabling _____ ______ to continue

Proofreading exonucleases decrease the chances of mismatched nucleotides from 1 in every 10⁵ to 1 in every _____. This is still short of the observed 1 in every 10¹⁰. The additional accuracy is due to the _______ ______ ______ process.

In the cell, both strands of the DNA duplex are replicated ________ at the _______ _____. What is required to create two template DNAs.

The junction between the newly separated template strands and the unreplicated duplex DNA is known as the ________ ______. It moves ________ toward the duplex region of ________ _____, leaving in its wake ____ _____ templates that each direct the synthesis of a ________ DNA strand

The _______ nature of DNA creates a complication for the simultaneous replication of the two exposed _______ at the ______ _____.

Why does the antiparallel nature of DNA create complications

As a result of the complication, on the template strand, the polymerase simply chases the moving ______ ______. The newly synthesized DNA strand directed by this template is known as the _______ ______.

Synthesis of the new DNA strand directed by the other _____ template is more _______. This template directs the DNA polymerase to move in the _______ direction of the replication fork. The new DNA strand directed by this template is nown as the ______ strand and must be synthesized in a _______ fashion

The ______ strand DNA polymerase can replicate its template as soon as it is exposed, but synthesis of the ______ strand must wait for movement of the replication fork to expose a ______ length of template before it can be replicated.

Each time a substantial length of new _______ strand is exposed, DNA synthesis is _______ and continues until it reaches the ___ end of the previous newly synthesized stretch of _____ strand DNA

The resulting short fragments of new DNA formed on the lagging strand are called _______ _______ and vary in length from 1000 to 2000 nucleotides in _______ and from 100 to 400 nucleotides in _______.

Shortly after being synthesized, Okazaki fragments are ________ joined together to generate a continuous, intact strand of new DNA. Therefore the fragments are ______ ______ in DNA replication

To accomplish covalent bonding of Okazaki fragments, the cell takes advantage of the ability of ___ _______ to do what DNA polymerase cannot, which is?

______ is a specialized ____ polymerase dedicated to making short ____ _____ on an _____ template.

The RNA primers made by primase are subsequently ______ by DNA polymerase. Although DNA polymerases incorporate only ___________ into DNA, they can initiate synthesis using either an ____ _____ or a ____ _____ annealed to the DNA template

Although both the leading and lagging strands require ______ to initiate DNA synthesis, the _______ of _______ function on the two strands is dramatically different

Each leading strand requires only a single _____ _____. In contrast, the discontinous synthesis of the _______ strand means that new primers are needed for each ______ _______.

Why is it that synthesis of the lagging strand can require hundreds of Okazaki fragments and their associated RNA primers?

Unlike the RNA polymerases involved in messenger RNA, ribosomal RNA and transfer RNA synthesis, primase does not require an ______ DNA sequence to initiate RNA synthesis. Instead, primases prefer to initiate RNA synthesis using an _____ template containing a particular ______ (___ in the case of E. coli primase.)

Primase activity is dramatically increased when it associates with another protein that acts at the replication fork called ____ ______. This protein unwinds the DNA at the _______ _____, creating an ssDNA template that can be acted on by _______.

The requirement for an ssDNA template and DNA helicase association ensures that ______ is only active the ______ _____.

To complete DNA replication, the _____ ______ used for initiation must be removed and replaced with _____. The process can be thought of as a DNA repair event

To replace the RNA primers with DNA, an enzyme called ______ recognizes and removes most of each RNA primer. This enzyme specifically ______ RNA that is base paired with _____.

Why does RNase H removes all of the RNA primer except the the ribonucleotide directly linked to the DNA end? How is the final ribonucleotide removed?

Removal of he RNA primer leaves a ____ in the dsDNA that is an ideal substrate for DNA polymerase. Which fills the _____ until every nucleotide is ____ ____, leaving a DNA molecule that is complete except for a break in the _______ _______ between the ______ & ________ of the repaired strand. This is referred to as a "_____"

The "nick" in the DNA can be repaired by an enzyme called _____ _______. The enzyme uses _____ _____ ______ (such as ____) to create a phosphodiester bond between an adjacent _________ and _____.

Only after all _______ are replaced by DNA and the associated _____ are sealed is DNA synthesis complete