Name: dna replication stress and fork stalling
Uploaded: 2020-11-23T11:47:35.000Z
Duration: 1 min 2 s
Channel: JoVE

K12 Standards

Alabama Course of Study Science

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.3

The DNA Replication Fork

בתא, שכפול דנ"א מתחיל תמיד במקומות ספציפיים בדנ"א הנקראים מקור השכפול.
בתחילת השכפול, מנחת המסוקים של הדנ"א האנזים נקשר ונע לאורך גדיל הדנ"א, משחרר ומפריד את הדנ"א. 
המבנה בצורת Y שנוצר על ידי גדילי הדנ"א הנפרדים נראה כמו מזלג בעל שני גוונים והופך לאתר של שכפול DNA; לכן, מבנה זה ידוע בשם "מזלג השכפול".
מולקולות דנ"א נפרדות וחד-גדיליות אלה נוטות ליצור לולאות דו-גדיליות או להריץ לאחור עם הגדיל השני. 
כדי למנוע זאת, חלבונים קושרי דנ"א חד-גדילי, או SSB, נקשרים לדנ"א חד-גדילי ומעכבים הרצה לאחור.
כעת הגדילים הבודדים החשופים של הדנ"א יכולים לשמש כתבניות לסינתזה של גדילי הבת המשלימים.

<ol>
	<li>מזוזי, עבד אל-גאני, ג'ורג'יה ולימזי וג'ואנה לואיזו. "מתח שכפול DNA: גורם, פתרון ומחלה." מחקר תאי ניסויי 329, מס '1 (2014): 85-93.</li>
	<li>סעדה, אניסיה אייט, שרה א. למברט ואנטוני מ. קאר. "שמירה על שלמות מזלג השכפול וכשירותו באמצעות מסלול הרקומבינציה ההומולוגית." תיקון DNA 71 (2018): 135-147.</li>
	<li>וואגה, שו וברוס סטילמן. "מזלג שכפול הדנ"א בתאים איקריוטים". סקירה שנתית של ביוכימיה (1998): 721-751.</li>
	<li>בודי, מייקל נ' ופול ראסל. "מחסום שכפול DNA". ביולוגיה נוכחית 11, מס '23 (2001): R953-R956.</li>
</ol>

מזלג שכפול ה-dna

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יש לשכפל את הגנום של האורגניזם בצורה יעילה וללא טעויות להתפתחותו והישרדותו. מזלג השכפול הוא אזור פעיל בצורת Y שבו שני גדילי DNA מופרדים ומשוכפלים ברציפות. הצימוד של פתיחת הDNA וסינתזת גדילים משלימים הוא תכונה אופיינית של מזלג שכפול. לאורגניזמים עם מעגל קטן של DNA, כגון E. coli, יש לעתים קרובות מקור יחיד של שכפול; לכן, יש להם רק שני מזלגות שכפול, אחד לכל כיוון מוביל הרחק ממיקום הפתח הראשוני. באורגניזמים בעלי גנום גדול, שכפול ה-DNA לא נעשה מנקודת מוצא בודדת אלא במזלגות שכפול מקומיים רבים ומובהקים.

ההתקדמות ללא הפרעה של מזלג השכפול הכרחית לשכפול ה-DNA המלא ויציבות הגנום; עם זאת, מזלג השכפול נעצר לעתים קרובות על ידי גורמים פנימיים או חיצוניים שיכולים להאט או לעצור את התקדמותו, וכתוצאה מכך ללחץ שכפול. מתח שכפול גורם לאי יציבות גנומית, שהיא סימן היכר של מחלות כמו סרטן. חוסר יציבות גנומית מאופיינת בשינויים גנומיים ותדירות מוגברת של מוטציות מזיקות. התנועה של מזלג השכפול יכולה להיעצר מכמה סיבות. לדוגמה, התרופה הידרוקסיאוריאה מדלדלת את מאגר הנוקלאוטידים הזמינים לשילוב בגדיל הבת, ומעכבת את מזלג השכפול. בעיות נוספות שעלולות לעכב את התקדמות מזלג השכפול כוללות נגעי DNA, התנגשות בין מזלג שכפול למתחם שעתוק, ופגמים באנזימים המעורבים בשכפול ה-DNA.

לתא יש מגוון מנגנוני תיקון כדי להניע מחדש את מזלג השכפול שנתקע. נקודות ביקורת של שלב S אינן מאפשרות לתאים להתחיל במיטוזה עד להשלמת תיקון ה-DNA. בנוסף, תחול מחדש של מזלג יכול להפעיל מחדש את סינתזת ה-DNA על ידי עקיפת נגע או בלוק DNA. למרות המנגנונים החזקים הללו, לפעמים לא ניתן להפעיל מחדש מזלגות שנתקעו, מה שמוביל להתמוטטות של מזלג, תוך עצירת שכפול ה-DNA.

</div>

מזלג שכפול ה-DNA

יש לשכפל את הגנום של אורגניזם בצורה יעילה ונטולת טעויות לצורך גדילתו והישרדותו. מזלג השכפול הוא בצורת Y ...

Education

JoVE Core

Molecular Biology

ביולוגיה מולקולרית

Unit 1

יחידה 1

DNA Replication

שכפול DNA

KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

dna replication stress and fork stalling

DNA replication stress and fork stalling can disrupt the copying of a cell’s genome. The replication fork is the Y-shaped site where DNA strands separate and new complementary strands are made at the same time. This linked unwinding and synthesis helps cells duplicate DNA efficiently and with fewer errors.

In organisms with small circular DNA, such as E. coli, replication often begins at a single origin. That setup creates two replication forks that move in opposite directions from the opening site. In organisms with large genomes, DNA copying starts at many local replication forks instead of one origin.

Smooth fork movement is needed for complete DNA replication and genome stability. When a fork slows or stops, the cell experiences replication stress. This stress can lead to genomic instability, which means more harmful mutations and other DNA changes. Genomic instability is also a hallmark of diseases such as cancer.

Several problems can stall a replication fork. Hydroxyurea is one example. It reduces the pool of nucleotides needed to build the daughter strand. DNA lesions, collisions between a replication fork and a transcription complex, and defects in DNA replication enzymes can also block fork progress.

Cells have repair systems that can restart stalled forks. S-phase checkpoints prevent cells from entering mitosis until DNA repair is finished. Fork repriming can also restart DNA synthesis by skipping over a DNA lesion or other block. Even so, some stalled forks cannot be restarted, and fork collapse stops DNA replication.

DNA Replication Stress and Fork Stalling

replication in prokaryotes

Prokaryotic DNA Replication Steps

Prokaryotic DNA replication copies the chromosome before a cell divides. In bacteria, this process starts at a specific DNA site called the origin of replication. From there, the cell builds new DNA strands using enzymes that match each base with its partner. The DNA double helix first opens so replication can begin. As the strands separate, each original strand serves as a template. New nucleotides are added in a set order, which helps the cell make an accurate copy of its genetic material. <br...

replication in eukaryotes

Eukaryotic DNA Replication Steps

Eukaryotic DNA replication copies the genome before a cell divides. In eukaryotes, this process happens in a controlled sequence so each new cell can receive genetic information. Replication begins at specific sites on the DNA called origins. The DNA is opened, and each original strand serves as a template, meaning it guides the building of a new complementary strand. This sets up two DNA molecules from one original molecule. The process then continues as enzymes add matching nucleotides to...

dna base pairing

DNA Base Pair Rules

DNA base pairing follows specific rules that help DNA carry genetic information. In DNA, the bases pair in a regular way, with adenine matching thymine and cytosine matching guanine. These pairs fit together because of their chemical shapes and bonding patterns. These base pairs also support accurate DNA replication. When DNA copies itself, each strand can guide the formation of a new partner strand using the pairing rules. Understanding base pairing helps explain how DNA stays organized and...

the dna replication fork

DNA replication stress and fork stalling can disrupt the copying of a cell’s genome. The replication fork is the Y-shaped site where DNA strands separate and new complementary strands are made at the same time. This linked unwinding and synthesis helps cells duplicate DNA efficiently and with fewer errors. In organisms with small circular DNA, such as E. coli, replication often begins at a single origin. That setup creates two replication forks that move in opposite directions from the opening...

proofreading

Proofreading in DNA Replication

Proofreading in DNA replication helps keep DNA copying accurate. It is a built-in check that reduces mistakes as new DNA is made. DNA polymerase is the enzyme that carries out this checking during replication. When it adds a wrong nucleotide, it can remove the error and replace it with the correct one. This correction step helps maintain the correct DNA sequence. Proofreading is an important part of DNA replication because it supports genetic accuracy. By catching mistakes as they happen, the...

dna helicases

Helicase Motor Proteins in DNA Unwinding

Helicase motor proteins help unwind DNA during key cell processes. They use energy from ATP hydrolysis, which is the breakdown of ATP to release usable energy. This movement lets helicases separate DNA strands when the cell needs access to the genetic code. These enzymes are involved in DNA replication, repair, recombination, and transcription. Helicases are found in all living organisms, but their structure, function, and movement can differ. In prokaryotes, the DnaB helicase moves along the...

the replisome

DNA Replication Teams in the Replisome

DNA replication depends on the replisome, a coordinated complex of proteins that copies DNA with high accuracy. This DNA replication machinery brings many parts together so the leading and lagging strands can be made at the same time. A key idea in this process is the trombone model, proposed by Bruce Alberts in 1980. In this model, a loop forms on the lagging strand when a primer is made. The loop grows as an Okazaki fragment is synthesized, then falls apart when that fragment is finished.

lagging strand synthesis

Okazaki Fragments in DNA Replication

DNA replication uses a leading strand and a lagging strand, and they are made at different rates. The leading strand starts first. The lagging strand starts later, moves more slowly, and is made in pieces. The two strands also differ in direction and starting steps. Leading strand synthesis follows the opening replication fork. Lagging strand synthesis goes in the opposite direction. The leading strand needs one RNA primer, while the lagging strand needs multiple RNA primers. After priming,...

mismatch repair

Fixing DNA Copying Errors

Mismatch repair helps fix DNA copying errors after replication. When DNA is copied, small mistakes can slip through. This repair system finds those mismatches and corrects them before they become permanent changes. The process depends on a group of mutator proteins. These proteins work together to detect the error, remove the incorrect section, and help replace it with the proper DNA sequence. In this way, mismatch repair supports the accuracy of genetic information. If the repair system does...

dna topoisomerases

How Topoisomerases Untwist DNA

DNA topoisomerases are enzymes that untwist DNA when it becomes overcoiled during cell processes such as DNA replication and transcription. They help regulate positive and negative DNA supercoiling without changing the nucleotide sequence. Clockwise overwinding creates positively supercoiled DNA, while counterclockwise underwinding produces negatively supercoiled DNA. Topoisomerases are grouped into two main types. Type I topoisomerases act on one strand of double-stranded DNA, and they...

telomeres and telomerase

How Telomerase Protects Chromosome Ends

Telomerase helps protect chromosome ends during eukaryotic DNA replication. When the final primer is removed, a single-stranded DNA fragment can remain at the end of the chromosome. That piece cannot be copied in the same way as the rest of the strand because there is no 3' end for new DNA to attach to. If this end piece is not replaced, chromosomal DNA is gradually lost each time a cell divides. The exposed single-stranded DNA can also trigger a DNA damage response from enzymes that detect it.

non-nuclear inheritance

Cytoplasmic Inheritance in Cells

Cytoplasmic inheritance explains how some traits are passed through cell structures outside the nucleus. These traits come from organelles such as mitochondria and chloroplasts. They do not follow the usual nuclear pattern of inheritance. Mitochondria and chloroplasts each contain their own DNA. Because of this, they can carry genetic information that affects cell function and certain visible traits. In high school biology, this topic helps explain why not all inherited traits come from...

animal mitochondrial genetics

Animal Mitochondrial DNA and Inheritance

Animal mitochondrial DNA has its own genome, separate from nuclear DNA. It is a double-stranded, closed circular molecule with about 20,000 base pairs. One strand is the heavy, or H-strand, because it is rich in guanine. The other is the light, or L-strand, because it is rich in cytosine. Compared with nuclear DNA, mitochondrial DNA has very little non-coding sequence. It also has no introns. Its genes are packed closely together, and some even overlap. The D-loop is the main non-coding...

comparing mitochondrial, chloroplast, and prokaryotic genomes

comparing-mitochondrial-chloroplast-and-prokaryotic-genomes

How Organelle Genomes Resemble Bacteria

Mitochondrial and chloroplast genomes still keep several traits from their bacterial ancestors. They also show changes that happened after these organelles evolved inside eukaryotic cells. Eukaryotic cells are cells with a nucleus, while prokaryotes are cells without one. Like prokaryotic genomes, mitochondrial and chloroplast genomes do not bind with histone-like proteins. They also do not show the complex chromosome-like packaging seen in eukaryotes. Mitochondria and chloroplasts divide by...

export of mitochondrial and chloroplast genes

export-of-mitochondrial-and-chloroplast-genes

How Organelles Pass Genes to the Nucleus

Eukaryotic cells can carry three genetic systems: nuclear, mitochondrial, and chloroplast. Over time, many genes from mitochondria and chloroplasts have moved into the nucleus. In some plant species, this gene transfer is still happening. This movement has affected large parts of the genome in some organisms. About 18% of the Arabidopsis thaliana nuclear genome is thought to come from the chloroplast's cyanobacterial ancestor. In yeast, around 75% of the genome is derived from the...

single-strand dna binding proteins

How SSB Proteins Protect DNA Strands

Single-strand DNA-binding proteins, or SSB proteins, help protect DNA during replication. When double-stranded DNA is unwound, the two strands separate. SSB proteins bind to the exposed single strands and keep them stable. These proteins bind in a sequence-independent manner. That means the order of the nitrogenous bases does not have to follow a specific pattern for SSB proteins to attach. Their binding straightens single-stranded DNA and makes it more rigid. This support may help DNA...

key terms and concepts

Learn how DNA replication forks can stall, why replication stress matters, and how cells restart DNA synthesis to protect genome stability.

DNA Replication Stress and Fork Stalling | JoVE K12

molecular biology

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9-12.B.1.2.a

DNA Replication Stress and Fork Stalling

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